Saturation mutagenesis of Thr862, the amino acid essential for substrate specificity of Eco57I restriction endonuclease

Abstract

Type IIG restriction endonuclease (RE) Eco57I cleaves DNA 16/14 nucleotides away from the asymmetric recognition sequence 5’-CTGAAG. The enzyme also possesses methyltransferase activity that modifies the second A base within the 5’-CTGAAG strand of the target duplex (underlined). In previous studies, Eco57I mutants with altered substrate specificity 5’-CTGRAG were isolated. These mutant enzymes have Asn or Ser instead of Thr in the 862th position of the protein. In order to evaluate the impact of T862 on the substrate specificity, it was changed to the other 17 amino acids. The in vivo cleavage activity and substrate specificity of the resulting mutant enzymes was examined (i) by testing lethality of the mutants to the host cells in the absence or presence of Eco57I (specificity 5’-CTGAAG) and GsuI (specificity 5’-CTGGAG) methyltransferases, and (ii) by testing the ability of the mutants to induce SOS DNA repair response in the absence or presence of protecting methyltransferases. The results indicate that mutants T862G, T862C and, probably, T862A and T862D could display altered substrate specificity. The recognition sequence of T862F, H, K, L, Q, M and Y mutants was the same as that of the wild type enzyme. The remaining substitutions rendered the enzyme catalytically inactive. Keywords: Eco57I restriction endonuclease, altered specificity, saturated mutagenesis, SOS induction