Selection of Non-specific DNA Cleavage Sites by the Type IC Restriction Endonuclease EcoR124I

Abstract The Type IC restriction endonuclease Eco R124I binds specifically to its recognition sequence but subsequently translocates non-specific DNA past the complex in an ATP-dependent mechanism. The enzyme thus has the potential to cleave DNA at loci distant from the recognition site. We have scrutinised the link between translocation and cleavage on linear and circular DNA substrates. On linear DNA carrying two recognition sites, the majority of cleavages at loci distant from the recognition site occurred between the two sites, regardless of the inter-site distance or relative orientations. On circular DNA carrying one site, distant cleavages occurred throughout the DNA but an equivalent linear molecule underwent considerably fewer cleavages at distant loci. These results agree with published models for DNA tracking. However, on every molecule investigated, discrete cleavage sites were also observed within ±250 bp of the recognition sites. The localised cleavages were not confined to particular DNA sequences and were independent of DNA topology. We propose a model to account for both distant and localised cleavage events. The conformation of the DNA loop extruded during tracking may result in two DNA segments being held in proximity to the restriction moiety on the protein, one close to the Eco R124I site and another distant from the site: cleavage may occur in either segment. Alternatively, the cutting of DNA close to recognition sites may be the result of multiple nicks being generated in the expanding loop before any extensive translocation.