SELECTION OF NONSPECIFIC DNA CLEAVAGE SITES BY THE TYPE IC RESTRICTION-ENDONUCLEASE ECOR124I

The Type IC restriction endonuclease EcoR124I binds specifically to it s 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 linea r 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 dist ance or relative orientations. On circular DNA carrying one site, dist ant cleavages occurred throughout the DNA but an equivalent linear mol ecule underwent considerably fewer cleavages at distant loci. These re sults agree with published models for DNA hacking. However, on every m olecule investigated, discrete cleavage sites were also observed withi n +/-250 bp of the recognition sites. The localised cleavages were not confined to particular DNA sequences and were independent of DNA topo logy. We propose a model to account for both distant and localised cle avage events. The conformation of the DNA loop extruded during hacking may result in two DNA segments being held in proximity to the restric tion moiety on the protein, one close to the EcoR124I site and another distant from the site: cleavage may occur in either segment. Alternat ively, the cutting of DNA close to recognition sites may be the result of multiple nicks being generated in the expanding loop before any ex tensive translocation. (C) 1997 Academic Press Limited.