Abstract
A closer inspection of the amino acid sequence of EcoP15I DNA methyltransferase revealed a region of similarity to the PDXn(D/E)XK catalytic site of type II restriction endonucleases, except for methionine in EcoP15I DNA methyltransferase instead of proline. Substitution of methionine at position 357 by proline converts EcoP15I DNA methyltransferase to a site-specific endonuclease. EcoP15I-M357P DNA methyltransferase specifically binds to the recognition sequence 5'-CAGCAG-3' and cleaves DNA asymmetrically EcoP151-M357P.DNA methyltransferase specifically binds to the recognition sequence 5'-CAGCAG-3' and cleaves DNA asymmetrically, 5'-CAGCAG(N)(10)-3', as indicated by the arrows, in presence of magnesium ions.
Highlights
Type II restriction endonuclease (REase) are homodimers that typically recognize a palindromic sequence of 4 – 8 bp and cut the DNA within or close to their recognition sequence in the presence of Mg2ϩ to give a 5Ј-phosphate and a 3Ј-hydroxyl end
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
The asymmetric nature of the EcoP15I recognition sequence is inconsistent with the use of a symmetric dimer for recognition and DNA cleavage, as found for the type IIP restriction endonucleases
Summary
Type II REases are homodimers that typically recognize a palindromic sequence of 4 – 8 bp and cut the DNA within or close to their recognition sequence in the presence of Mg2ϩ to give a 5Ј-phosphate and a 3Ј-hydroxyl end. In M.EcoP15I, a PDXn(D/E)XK-like motif is present in which the partially conserved proline is replaced by methionine 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]. We have not found any MTases in the Protein Data Bank containing the Mg2ϩ binding motif that is present in M.EcoP15I
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