Identification and Mutational Analysis of Mg2+ Binding Site in EcoP15I DNA Methyltransferase

Abstract

EcoP15I DNA methyltransferase catalyzes the transfer of the methyl group of S-adenosyl-L-methionine to the N 6 position of the second adenine within the doublestranded DNA sequence 5-CAGCAG-3. To achieve catalysis, the enzyme requires a magnesium ion. Binding of magnesium to the enzyme induces significant conformational changes as monitored by circular dichroism spectroscopy. EcoP15I DNA methyltransferase was rapidly inactivated by micromolar concentrations of ferrous sulfate in the presence of ascorbate at pH 8.0. The inactivated enzyme was cleaved into two fragments with molecular masses of 36 and 35 kDa. Using this affinity cleavage assay, we have located the magnesium bindinglike motif to amino acids 355-377 of EcoP15I DNA methyltransferase. Sequence homology comparisons between EcoP15I DNA methyltransferase and other restriction endonucleases allowed us to identify a PD(X) n (D/E)XK-like sequence as the putative magnesium ion binding site. Point mutations generated in this region were analyzed for their role in methyltransferase activity, metal coordination, and substrate binding. Although the mutant methyltransferases bind DNA and S-adenosyl-L-methionine as well as the wild-type enzyme does, they are inactive primarily because of their inability to flip the target base. Collectively, these data are consistent with the fact that acidic amino acid residues of the region 355-377 in EcoP15I DNA methyltransferase are important for the critical positioning of magnesium ions for catalysis. This is the first example of metal-dependent function of a DNA methyltransferase. These findings provide impetus for exploring the role(s) of metal ions in the structure and function of DNA methyltransferases. DNA methyltransferases (MTases)1 sequence-specifically modify DNA in a wide range of organisms. The enzymes require the methyl donor S-adenosyl-L-methionine (AdoMet) to modify their target base (1). Three classes of DNA MTases, namely, the C 5 -cytosine MTases, N 4 -cytosine MTases, and N 6 -adenine-specific MTases have been identified on the basis of sequence homology and type of methylation catalyzed (2, 3). Structure-guided sequence comparison analysis revealed that all three classes of MTase are closely related to one another (4, 5). EcoP15I DNA methyltransferase (M.EcoP15I) adds a methyl group to the second adenine in the recognition sequence 5Ј-CAGCAG-3Ј in the presence of AdoMet (6). It is an N 6 -adenine MTase, and like all N 6 -adenine MTases, M.EcoP15I contains two highly conserved sequences, FXGXG (motif I) at position 444 -448 and DPPY (motif IV) at position 125-128 (7). Although mutations in motif I completely abolished AdoMet binding but left target DNA recognition unaltered, mutations in motif IV resulted in loss of enzyme activity, but AdoMet and DNA binding were not affected (8). By employing chemical modification using thiol-directed agents and site-directed mutagenesis, it was demonstrated that cysteine 344 in M.EcoP15I was necessary for enzyme activity and played an essential role in DNA binding (9). W...

Kinetics of Methylation by EcoP1I DNA Methyltransferase

“…Mutations in the metal-binding motif, amino acid residues 355–377 in M.EcoP15I, resulted in a loss of methylation activity. It was shown that the Mg 2+ ion was required for base flipping during the methyl transfer reaction [8]. This study reports the order of substrate binding, two metal ion requirement, and mode of methylation by EcoP1I DNA MTase.…”

“…Mutational analysis of the AdoMet-binding motif and the catalytic motif of M.EcoP1I abolished methylation activity suggesting the importance of the motifs in AdoMet binding and transfer of the methyl group to DNA [7]. Unlike M.EcoP1I, EcoP15I DNA MTase (M.EcoP15I), the other well-characterized-MTase from the Type III R-M system, methylates double-strand DNA in the presence of Mg 2+ and does not methylate single-strand DNA [8]. Mutations in the metal-binding motif, amino acid residues 355–377 in M.EcoP15I, resulted in a loss of methylation activity.…”

A Mutation in the Mod Subunit of EcoP15I Restriction Enzyme Converts the DNA Methyltransferase to a Site-specific Endonuclease

“…The sequence of primer 2 was designed to change the methionine 357 to alanine and to create a restriction site (PvuII). A single strand DNA template containing uracil residues was prepared for E. coli strain CJ 236 that harbored pGEM3Zf(Ϫ)M.EcoP15I (11). Primers 1 and 2 were hybridized separately to the single-stranded DNA, and oligonucleotidedirected mutagenesis was performed as described previously (14).…”

“…Using a Fenton chemistry affinity cleavage assay, we identified the magnesium binding-like motif as amino acids 355-377 (11). Sequence homology comparisons between EcoP15I DNA MTase and other restriction endonucleases allowed us to identify a PDX n (D/E)XK-like sequence as the putative magnesium ion binding site (Fig.…”

“…Mutational analysis of this region and detailed biochemical characterization of mutant MTases are consistent with the fact that acidic amino acid residues of this region 355-377 in M.EcoP15I are important for the critical positioning of magnesium ions for catalysis. This is the first example of a metaldependent function for a DNA methyltransferase (11 …”