SATURATION MUTAGENESIS OF HIS(114) OF ECORI REVEALS RELAXED-SPECIFICITY MUTANTS

EcoRI recognizes and cleaves DNA at GAATTC sites and is one of the bes t characterized sequence-specific restriction endonucleases (ENases). In previous studies, an EcoRI mutant, which exhibited relaxed substrat e specificity and cleaved both canonical and EcoRI star sites, was iso lated. This mutant enzyme has Tyr instead of His(114). Here, we subjec ted residue 114 of the EcoRI ENase to saturation mutagenesis. The resu lting mutant enzymes were characterized both in vivo and in vitro, res ulting in the identification of mutants with canonical (H114K, Q, D, I ) or relaxed (K114Y, F, S, T) specificity, as well as one mutant with severely impaired activity (H114P). In the X-ray structure of an EcoRI substrate complex, His(114) is located between the catalytic and recog nition regions of EcoRI and may directly contact the DNA phosphate bac kbone. Based on our genetic and biochemical findings and the X-ray str ucture, we propose that His(114) participates in substrate recognition and catalysis, either directly, via protein-DNA interactions, or indi rectly, by mediating conformational changes that trigger DNA cleavage in response to substrate recognition.