Structural and Functional Analysis of the Symmetrical Type I Restriction Endonuclease R.EcoR124I_NT

<div><p>Type I restriction-modification (RM) systems are comprised of two multi-subunit enzymes, the methyltransferase (∼160 kDa), responsible for methylation of DNA, and the restriction endonuclease (∼400 kDa), responsible for DNA cleavage. Both enzymes share a number of subunits. An engineered RM system, EcoR124I_NT, based on the N-terminal domain of the specificity subunit of EcoR124I was constructed that recognises the symmetrical sequence GAAN₇TTC and is active as a methyltransferase. Here, we investigate the restriction endonuclease activity of R. EcoR124I_NT<em>in vitro</em> and the subunit assembly of the multi-subunit enzyme. Finally, using small-angle neutron scattering and selective deuteration, we present a low-resolution structural model of the endonuclease and locate the motor subunits within the multi-subunit enzyme. We show that the covalent linkage between the two target recognition domains of the specificity subunit is not required for subunit assembly or enzyme activity, and discuss the implications for the evolution of Type I enzymes.</p>