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

Abstract

A closer inspection of the amino acid sequence of EcoP15I DNA methyltransferase revealed a region of similarity to the PDX n (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-M357P⅐DNA methyltransferase specifically binds to the recognition sequence 5-CAGCAG-3 and cleaves DNA asymmetrically, 5-CAG-CAG(N) 10 -3, as indicated by the arrows, in presence of magnesium ions.Sequence discrimination by DNA-binding proteins is one of the most remarkable examples of molecular recognition in biochemistry. Restriction-modification (R-M) 4 systems are paradigms for the study of protein-DNA recognition. More than 3600 R-M enzymes have been identified so far, and they manifest 218 unique recognition specificities (1). These enzymes represent the largest family of functionally related enzymes. R-M systems comprise pairs of intracellular enzymatic activities; that is, restriction endonuclease (REase) activity and DNA methyltransferase (MTase) activity. The REase recognizes specific sequences and catalyzes cleavage of double-stranded DNA. The MTase prevents the REase from digesting endogenous DNA by modifying the same target sequence with the methyl group from S-adenosyl-L-methionine (AdoMet) (2). R-M enzymes are classified into types I, II, III, and IV, based on their recognition sequence, subunit composition, cleavage position, and cofactor requirements (3). The simplest R-M systems are the type II enzymes. These generally consist of two separate enzymes, one responsible for restriction and the other for modification of the cognate DNA sequences. The orthodox 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 Mg 2ϩ to give a 5Ј-phosphate and a 3Ј-hydroxyl end. Although thousands of type II REases are known, the structures of only 19 have been reported to date (4). Structural and mutational analysis of several type II REases reveal that the sequence PDX n (D/E)XK (n ϭ 1-30 residues) acts as a catalytic/Mg 2ϩ binding signature motif (5). For the great majority of type II REases, Mg 2ϩ is an essential cofactor that can be substituted with Mn 2ϩ . The essential divalent ion cofactor is bound to the two carboxylates and the main carbon of the hydrophobic residue X of the PDX n (D/E)XK motif as well as to one of the non-bonding oxygens of the phosphate to be attacked. The fact that many REases have a common catalytic sequence motif suggests that they follow the same mechanism with at least one obvious exception, BfiI restriction endonuclease, which does not require a divalent metal ion cofactor (6).Type III R-M systems are multisubunit and multifunctional enzymes that exhibit bo...