The T4 dam' gene has been cloned (S. L. Schiagman and S. Hattman, Gene 22:139-156, 1983)

A mutant (designated mec-) of Escherichia coli F+ 100 endo 1-su+ rK -mK+ has been isolated which is defective in cytosine-specific deoxyribonucleic acid (DNA) methylase activity. The DNA of this mutant, as well as the DNA of phages A and fd propagated in it, is virtually devoid of 5-methvl-cytosine (MeC); in contrast, the mutation has no significant effect on the level of N6-methyladenine in DNA.Phage A grown on the mec-mutant is more strongly restricted by N-3-containing cells than is A grown on the mec+ parent. These results suggest that methylation of certain cytosine residues by the E. coli K-12 enzyme partially protects A DNA from either the N-3 restriction nuclease or against secondary degradation subsequent to N-3-specific degradation. Analysis of the MeC level in viral and cellular DNA obtained from mec+, mec+ (mNI+), and mec-(mN3+) strains has led to the conclusion that the R-factor controlled DNA-cytosine methylase may be capable of methylating a sequence(s) which is a substrate for the K-12 enzyme.

The bacteriophage T4 dam gene, encoding the Dam DNA [N6-adenine]methyltransferase (MTase), has been subcloned into the plasmid expression vector, pJW2. In this construct, designated pINT4dam, transcription is from the regulatable phage lambda pR and pL promoters, arranged in tandem. A two-step purification scheme using DEAE-cellulose and phosphocellulose columns in series, followed by hydroxyapatite chromatography, was developed to purify the enzyme to near homogeneity. The yield of purified protein was 2 mg/g of cell paste. The MTase has an s20,w of 3.0 S and a Stokes radius of 23 A and exists in solution as a monomer. The Km for the methyl donor, S-adenosylmethionine, is 0.1 x 10(-6) M, and the Km for substrate nonglucosylated, unmethylated T4 gt- dam DNA is 1.1 x 10(-12) M. The products of DNA methylation, S-adenosyl-L-homocysteine and methylated DNA, are competitive inhibitors of the reaction; Ki values of 2.4 x 10(-6) M and 4.6 x 10(-12) M, respectively, were observed. T4 Dam methylates the palindromic tetranucleotide, GATC, designated the canonical sequence. However, at high MTase:DNA ratios, T4 Dam can methylate some noncanonical sequences belonging to GAY (where Y represents cytosine or thymine).

DNA-adenine methylation at certain GATC sites plays a pivotal role in bacterial and phage gene expression as well as bacterial virulence. We report here the crystal structures of the bacteriophage T4Dam DNA adenine methyltransferase (MTase) in a binary complex with the methyl-donor product S-adenosyl-L-homocysteine (AdoHcy) and in a ternary complex with a synthetic 12-bp DNA duplex and AdoHcy. T4Dam contains two domains: a seven-stranded catalytic domain that harbors the binding site for AdoHcy and a DNA binding domain consisting of a five-helix bundle and a β-hairpin that is conserved in the family of GATC-related MTase orthologs. Unexpectedly, the sequence-specific T4Dam bound to DNA in a nonspecific mode that contained two Dam monomers per synthetic duplex, even though the DNA contains a single GATC site. The ternary structure provides a rare snapshot of an enzyme poised for linear diffusion along the DNA.DNA MTases catalyze methyl group transfer from donor S-adenosyl-L-methionine (AdoMet), producing S-adenosyl-L-homocysteine (AdoHcy) and methylated DNA. Although most prokaryote DNA MTases are components of restriction-modification systems, some MTases are not associated with cognate restriction enzymes, such as the Escherichia coli DNA adenine MTase (Dam). This enzyme methylates an exocyclic amino nitrogen (N6) of the adenine in GATC 1,2 . Dam MTase gene orthologs are widespread among enteric bacteria and their bacteriophages (see Fig. 1 legend). Dam methylation is not essential for the viability of E. coli, unless it is combined with certain other mutations 3 ; however, Dam is an essential gene in Vibrio cholerae and Yersinia pesudotuberculosis, at least under the tested growth conditions 4 . Dam methylation is essential for the virulence of Salmonella serovar typhimurium in a murine model of typhoid fever [5][6][7] . These observations COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. NIH Public Access RESULTS Overall structure of DamThe monomeric Dam structure contains 9 β-strands and 11 α-helices (Fig. 1). The Nterminal region (residues 1-52) and C-terminal region (residues 149-259) come together, forming the catalytic domain (green in Figs. 1 and 2a) with a seven-stranded β-sheet, a characteristic feature of the class-I AdoMet-dependent MTases 28 . The N-terminal helices αZ and αA are located on one side of the β-sheet, and helices αC, αD1 and αD2 and αE are on the other side. Between strands β2 and β3 is a separate domain, which we designate as the target-recognition domain (TRD). TRD is composed of a five-helix bundle (αB1-αB5) (yellow in Figs. 1 and 2a) and a 25-residue segment containing a β-hairpin (β8 and β9) inserted between helices αB4 and αB5 (red). The overall dimensions of the molecule are 55 × 34 × 28 Å, with an open cleft located between the catalytic domain (green) and the TRD (yellow and red). Conserved residues and the effects of mutationsBased on the linear arrangement of conserved motifs in DNA MTases, T4Dam is classified in the...

The fluorescence of 2-aminopurine ((2)A)-substituted duplexes (contained in the GATC target site) was investigated by titration with T4 Dam DNA-(N6-adenine)-methyltransferase. With an unmethylated target ((2)A/A duplex) or its methylated derivative ((2)A/(m)A duplex), T4 Dam produced up to a 50-fold increase in fluorescence, consistent with (2)A being flipped out of the DNA helix. Though neither S-adenosyl-L-homocysteine nor sinefungin had any significant effect, addition of substrate S-adenosyl-L-methionine (AdoMet) sharply reduced the Dam-induced fluorescence with these complexes. In contrast, AdoMet had no effect on the fluorescence increase produced with an (2)A/(2)A double-substituted duplex. Since the (2)A/(m)A duplex cannot be methylated, the AdoMet-induced decrease in fluorescence cannot be due to methylation per se. We propose that T4 Dam alone randomly binds to the asymmetric (2)A/A and (2)A/(m)A duplexes, and that AdoMet induces an allosteric T4 Dam conformational change that promotes reorientation of the enzyme to the strand containing the native base. Thus, AdoMet increases enzyme binding-specificity, in addition to serving as the methyl donor. The results of pre-steady-state methylation kinetics are consistent with this model.