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

Abstract

The role of two sequence motifs (SM) as putative cleavage catalytic centers 77PDX13EAK (SM I) and 811PDX 20DQK (SM II) of type IV restriction endonuclease Eco57I was studied by site-directed mutational analysis. Substitutions within SM I; D78N, D78A, D78K, and E92Q reduced cleavage activity of Eco57I to a level undetectable both in vivo and in vitro. Residual endonucleolytic activity of the E92Q mutant was detected only when the Mg2+ in the standard reaction mixture was replaced with Mn2+. The mutants D78N and E92Q retained the ability to interact with DNA specifically. The mutants also retained DNA methylation activity of Eco57I. The properties of the SM I mutants indicate that Asp 78 and Glu92 residues are essential for cleavage activity of the Eco57I, suggesting that the sequence motif 77PDX 13EAK represents the cleavage active site of this endonuclease. Eco57I mutants containing single amino acid substitutions within SM II (D812A, D833N, D833A) revealed only a small or moderate decrease of cleavage activity as compared with wild-type Eco57I, indicating that the SM II motif does not represent the catalytic center of Eco57I. The results, taken together, allow us to conclude that the Eco57I restriction endonuclease has one catalytic center for cleavage of DNA.