DNA Adenine Methylase Research Articles

Correlation of DNA adenine methylase activity with spontaneous mutability in Escherichia coli K-12

Using a multicopy plasmid in which the tac promoter has been placed in front of the dam gene of Escherichia coli K-12, we show that levels of DNA adenine methylase activity are correlated with the spontaneous mutation frequency.

Cloning in Streptococcus pneumoniae of the gene for DpnII DNA methylase.

The gene coding for the pneumococcal DNA adenine methylase that recognizes the sequence 5'-GATC-3' was cloned in a strain of Streptococcus pneumoniae that lacked both restriction endonucleases DpnI and DpnII. The gene was cloned as a 3.7-kilobase fragment of chromosomal DNA from a DpnII-containing strain inserted in both possible orientations in the multicopy plasmid vector pMP5 to give recombinant plasmids pMP8 and pMP10. Recombinant plasmids were selected by their resistance to DpnII cleavage. Cells carrying the recombinant plasmids modified phage in vivo so that it was restricted by DpnI- but not DpnII-containing hosts. They also showed levels of DNA methylase activity five times higher than that in cells of the original DpnII strain. No DpnII activity was observed in the clones; therefore, it was concluded that the insert did not contain an intact DpnII endonuclease gene and that methylation of host DNA did not turn on a latent form of the gene.

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.

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.

Molecular cloning of a functional dam+ gene coding for phage T4 DNA adenine methylase

Phages T2 and T4 induce synthesis of a DNA-adenine methylase which is coded for by a phage gene, dam +. These enzymes methylate adenine residues in specific sequences which include G-A-T-C, the methylation site of the host Escherichia coli dam + methylase. Methylation of G-A-T-C to G-m 6A-T-C protects the site against cleavage by the MboI restriction nuclease. We have taken advantage of this property to enrich and screen for transformants which contain a cloned, functional T4 dam + gene. These recombinant molecules consist of a 1.85-kb HindIII fragment inserted into the plasmid pBR322; both orientations of the fragment express the methylase gene, suggesting that transcription is from a T4 promoter. We have tested the 1.85-kb insert for sensitivity to a variety of restriction nucleases and have found single sites for EcoRI, BalI, XbaI, and at least two sites for Bst/NI ( EcoRII). The relative positions of these restriction sites have also been determined. Physical mapping was carried out by Southern blot hybridization with 32P-labeled (nick-translated clone) probe. These experiments showed that the insert corresponds to a HindIII fragment located on the physical map of T4 between positions 16.2 and 18.1 kb from the T4 r-IIA- rIIB junction. E. coli dam − possesses several phenotypic differences from the wild-type dam + parent, including an increased sensitivity to 2-aminopurine (2-AP). We found that T4 dam + clones could relieve dam − cells of their increased sensitivity to 2-AP.

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.

Phenotypic reversal in dam mutants of Escherichia coli K-12 by a recombinant plasmid containing the dam+ gene.

A recombinant plasmid, pMQ3, carrying the dam gene of Escherichia coli K-12, was constructed and transformed into dam+ and dam- strains. Both dam- and dam+ strains containing pMQ3 showed a wild phenotype for all traits, including mutation rate, except for a 10-fold increase in DNA adenine methylase activity.

DNA methyltransferase-dependent transcription of the phage Mu mom gene.

The phage Mu mom gene controls an unusual DNA modification. Expression of the mom function requires an active host (dam+) DNA adenine methylase [S-adenosyl-L-methionine:DNA (6-aminopurine)-methyltransferase]; in dam- hosts, Mu development is normal except that the viral DNA does not undergo the mom modification. The present communication compares transcription of the mom gene in dam+ versus dam- cells. 32P-labeled probes were prepared by nick-translation of a purified mom gene-containing restriction fragment and of virion DNA, respectively. These probes were hybridized with various RNAs blotted onto nitrocellulose filters (after fractionation by agarose gel electrophoresis). The salient findings are: (i) mom-specific RNA was readily detected in dam+ lysogenic cells, but only after induction of the Mu prophage; (ii) the level of mom RNA was decreased at least to 1/20th in induced dam- Mu lysogens; and (iii) little difference, if any, was observed between dam+ and dam- cells with respect to total Mu transcripts produced after prophage induction. These results are in accord with the known pattern of mom gene expression and Mu development. They show that the host (dam+) DNA adenine methylase activity is required for transcription of the mom gene. This represents a unique example where a DNA methylase exerts a positive regulatory role in mRNA transcription; alternative mechanisms for this process will be discussed.

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.

Characterization of DNA adenine methylation mutants of Escherichia coli K12

The phenotypic traits of 7 independently isolated dam mutants of Escherichia coli have been examined. The mutant strains differ from the wildtype in the following respects: (1) decreased DNA adenine methylase activity in vivo and in vitro; (2) a 14–85-fold increase in spontaneous mutability; (3) decreased survival after ultraviolet irradiation; (4) a 10–21-fold increase in spontaneous induction of λ phage from lysogens; (5) a 3–17-fold increase in the level of recombination; and (6) inviability of double mutants containing dam − and 3itrecB − or recC −. Unmethylated fd phage chromosomes are able to replicate normally in dam − mutants. A mutant strain in which the dcm gene is deleted is viable, showing that the dcm gene product is dispensible for growth.

Studies on the substrate specificity of the DNA methylase activity fromEscherichia 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 the P1 modification methylase (M· Eco P1) and the DNA methylase (M· Eco dam) controlled by the Escherichia coli dam gene

Labeled oligonucleotides have been fractionated from pancreatic DNase digests of DNA that had been methylated in vitro with the P1 modification enzyme (M· Eco P1) or with the DNA-adenine methylase (M· Eco dam) controlled by the Escherichia coli dam gene. The sequences of methylated oligonucleotides were established for M· Eco dam modification of calf thymus DNA. The results show that M· Eco dam inethylates adenine residues contained in the twofold symmetrical sequence, 5′ … G-A-T-C … 3′. The sequence for the site methylated by M· Eco P1 has also been deduced; we propose that M· Eco P1 modification produces the following methylated pentameric sequence: 5′ … A-G-A ∗-C-Py … 3′ (where A ∗ = N 6 methyladenine and Py is C or T).

In vitro methylation of bacteriophage λ DNA by wild type ( dam +) and mutant ( damh) forms of the phage T2 DNA adenine methylase

The wild-type ( dam + ) and mutant ( dam h) forms of the bacteriophage T2 DNA adenine methylase have been partially purified; these enzymes methylate the sequence, 5/t' … G-A-Py … 3′ (Hattman et al., 1978 a). However, in vitro methylation studies using phage λ DNA revealed the following: (1) T2 dam + and dam h enzymes differ in their ability to methylate λ DNA; under identical reaction conditions the T2 dam h enzyme methylated λ DNA to a higher level than did the dam + enzyme. However, the respective methylation sites are equally distributed on the l and r strands. (2) Methylation with T2 dam h, but not T2 dam + protected λ against P1 restriction. This was demonstrated by transfection of Escherichia coli (P1) spheroplasts and by cleavage with R· EcoP1. (3) T2 dam + and dam h were similarly capable of methylating G-A-T-C sequences on λ DNA; e.g. λ· dam 3 DNA (contains no N 6-methyladonine) methylated with either enzyme was made resistant to cleavage by R· DpnII. In contrast, only the T2 dam h modified DNA was resistant to further methylation by M· EcoP1 (which methylates the sequence 5′ … A-G-A-C-Py … 3′; Hattman et al., 1978 b). (4) λ· dam 3 DNA was partially methylated to the same level with T2 dam + or T2 dam h; the two enzymes produced different patterns of G-A-C versus G-A-T methylation. We propose that the T2 dam + enzyme methylates G-A-C sequences less efficiently than the T2 dam h methylase; this property does not entirely account for the large difference in methylation levels produced by the two enzymes.

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 N 6-methyldeoxyadenosine. From this analysis we conclude that both T2 dam + and T2 dam h contain the sequence 5′…G-mA -Py…3′.

Location of the DNA-adenine methylase gene on the genetic map of phage T2.

The location of the gene controlling DNA-adenine methylase activity is reported to be in the region between genes 49 and T1 on the T2 genetic map. A new nomenclature is proposed to describe the various phage types affected by mutations in the methylase gene.