Purification and properties of theEco57lrestriction endonuclease and methylas—prototypes of a new class (type IV)

Abstract

The Eco57I restriction endonuclease and methylase were purified to homogeneity from the E.coli RR1 strain carrying the eco57IRM genes on a recombinant plasmid. The molecular weight of the denaturated methylase is 63 kDa. The restriction endonuclease exists in a monomeric form with an apparent molecular weight of 104-108 kDa. R.Eco57I also possesses methylase activity. The methylation activities of both enzymes modify the outer A residue in the target sequence 5'CTGAAG yielding N6-methyladenine. M.Eco57I modifies both strands of the substrate while R.Eco57I modifies only one. Only the methylase enzyme is stimulated by Ca2+. The restriction endonuclease shows an absolute requirement for Mg2+ and is stimulated by AdoMet. ATP has no influence on either activity of the enzymes. The subunit structure and enzymatic properties of the Eco57I enzymes distinguish them from all other restriction-modification enzymes that have been described previously. Therefore, RM.Eco57I may be regarded as a representative of a novel class of restriction-modification systems, and we propose to classify it as type IV.

Many Type IIs Restriction Endonucleases Interact with Two Recognition Sites before Cleaving DNA

“…These enzymes recognize short DNA sequences, 4 -8 bp long and cleave both strands of the DNA at fixed locations in or near their recognition sites (2). With one exception, BfiI (3), they require Mg 2ϩ or a similar divalent metal ion to cleave DNA (4), although a few also require AdoMet 1 for maximal activity (5). Many of the type II enzymes are homodimeric proteins that interact symmetrically with palindromic DNA sequences, so that one active site in the dimer is placed to cleave one strand of the DNA and the other active site the equivalent phosphodiester bond in the opposite strand: for example, EcoRV, BamHI, and BglI (6 -8).…”

Mutational Analysis of Two Putative Catalytic Motifs of the Type IV Restriction Endonuclease Eco57I

“…Structure-Function Organization of Eco57I-The restriction endonuclease Eco57I combines, in a single polypeptide DNAspecific recognition, cleavage, and methylation activities (4). This study provides evidence that the catalytic DNA cleavage center is located near the N-terminal end of Eco57I (Fig.…”

“…Restrictionmodification enzymes are traditionally divided into three classes designated type I, II, and III on the basis of enzyme subunit composition, cofactor requirements, substrate specificity characteristics, and reaction products (2). An increasing number of restriction endonucleases that do not fit into the conventional classification however have been reported (3)(4)(5)(6)(7). Their differences from the type I and type III enzymes are so substantial that a classification as new kinds of restriction endonucleases; type IIS, type IIT, type IV, and Bcg-like has been suggested (3)(4)(5)(6)(7)(8).…”

“…The type IV restriction endonuclease Eco57I has been studied in detail (4). Similar to type IIS endonucleases, it recognizes an asymmetric nucleotide sequence, cleaves both DNA strands outside the target site 5Ј-CTGAAG(N) 16/142 and exists in solution as a monomer.…”

Lactic Acid Bacteria Resistance to Bacteriophage and Prevention Techniques to Lower Phage Contamination in Dairy Fermentation

“…For that reason as well as from the evolutionary point of view, the type IV RM system is worth mentioning. A fusion of genes coding for Mod and Res subunits of type III systems was a key step for evolution of type IV RM [56]. The resulting endonuclease (revealing also methyltransferase activity) has an asymmetrical recognition sequence and cleavage occurs at a fixed distance from the recognition site, like for the type IIS enzymes.…”