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.     

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.     

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.     

Transient overexpression of DNA adenine methylase enables efficient and mobile genome engineering with reduced off-target effects

Homologous recombination of single-stranded oligonucleotides is a highly efficient process for introducing precise mutations into the genome of E. coli and other organisms when mismatch repair (MMR) is disabled. This can result in the rapid accumulation of off-target mutations that can mask desired phenotypes, especially when selections need to be employed following the generation of combinatorial libraries. While the use of inducible mutator phenotypes or other MMR evasion tactics have proven useful, reported methods either require non-mobile genetic modifications or costly oligonucleotides that also result in reduced efficiencies of replacement. Therefore a new system was developed, Transient Mutator Multiplex Automated Genome Engineering (TM-MAGE), that solves problems encountered in other methods for oligonucleotide-mediated recombination. TM-MAGE enables nearly equivalent efficiencies of allelic replacement to the use of strains with fully disabled MMR and with an approximately 12- to 33-fold lower off-target mutation rate. Furthermore, growth temperatures are not restricted and a version of the plasmid can be readily removed by sucrose counterselection. TM-MAGE was used to combinatorially reconstruct mutations found in evolved salt-tolerant strains, enabling the identification of causative mutations and isolation of strains with up to 75% increases in growth rate and greatly reduced lag times in 0.6 M NaCl.     

Proximal recognition sites facilitate intrasite hopping by DNA adenine methyltransferase: mechanistic exploration of epigenetic gene regulation

The methylation of adenine in palindromic 5'-GATC-3' sites by Escherichia coli Dam supports diverse roles, including the essential regulation of virulence genes in several human pathogens. As a result of a unique hopping mechanism, Dam methylates both strands of the same site prior to fully dissociating from the DNA, a process referred to as intrasite processivity. The application of a DpnI restriction endonuclease-based assay allowed the direct interrogation of this mechanism with a variety of DNA substrates. Intrasite processivity is disrupted when the DNA flanking a single GATC site is longer than 400 bp on either side. Interestingly, the introduction of a second GATC site within this flanking DNA reinstates intrasite methylation of both sites. Our results show that intrasite methylation occurs only when GATC sites are clustered, as is found in gene segments both known and postulated to undergo in vivo epigenetic regulation by Dam methylation. We propose a model for intrasite methylation in which Dam bound to flanking DNA is an obligate intermediate. Our results provide insights into how intrasite processivity, which appears to be context-dependent, may contribute to the diverse biological roles that are carried out by Dam.     

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′.     

In vitro methylation of bacteriophage lambda 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., 1978a). 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·dam₃ DNA (contains no N⁶-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., 1978b). (4) λ·dam₃ 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.     

Delayed methylation and the matrix bound DNA methylase

It is shown that the methylation of DNA that occurs in isolated nuclei is "delayed methylation". This methylation is not reduced in nuclei which have been pretreated with 0.2M NaCl to extract the soluble methylase suggesting that this methylation is the product of a firmly bound matrix associated DNA methylase. Evidence is provided that, like the methylase, the DNA substrate is associated with the nuclear matrix.     

The unusual conformation adopted by the adenine tracts in kinetoplast DNA

To determine the structural features responsible for the curvature of kinetoplast DNA, we studied 13 adenine tracts in Crithidia fasciculata kinetoplast DNA. The structures of the A tracts were analyzed by cutting the DNA with hydroxyl radical. Reactivity of hydroxyl radical toward the DNA backbone progressively decreased in the 5'----3' direction of each A tract. The cutting pattern of the T-rich strand was offset by 1 or 2 bp from the pattern on the A-rich strand. An A tract in a restriction fragment from plasmid pBR322 had the same cutting pattern as the kinetoplast A tracts. We interpret these experiments to show that in A tracts the width of the minor groove decreases smoothly from the 5'----3' end of the A tract.     

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.     

Structural and functional insights into the mechanism by which MutS2 recognizes a DNA junction

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