Functional consequences of mutating conserved SF2 helicase motifs in the Type III restriction endonuclease EcoP15I translocase domain

► We mutated helicase motifs predicted in Type III restriction enzyme EcoP15I. ► We examined functional consequences on EcoP15I enzyme activity upon CD spectroscopy. ► Classical helicase motifs I-VI are important for ATP and DNA hydrolysis by EcoP15I. ► Newly assigned motifs Q-tip, Ia and Va are not essential for activity of EcoP15I. ► DNA binding is only marginally reduced (2-7 fold) in all variants tested.