Analysis of the sliding process of restriction enzymes by single molecule techniques

Type II restriction enzymes slide along the DNA in the process of searching for their recognition site. This is a random movement, interrupted by 3D excursions ("jumps") [1,2]. It is not yet clear, whether the sliding process of Type II restriction enzymes along the DNA double strand follows the pitch of the double helix or not. The spatial resolution to detect fluorescently labeled restriction enzymes is not good enough to answer this question. For this purpose it is planned to produce fusions of selected homodimeric restriction enzymes with long extensions consisting of quasi-linear protein structures that are fluorescently labeled at their distal ends. This should allow following the movement of the widely separated fluorescent labels by TIRFM (total internal reflection fluorescence microscopy) with superior spatial resolution and thereby to decide whether restriction enzymes follow the helical geometry of the DNA. To this end, we started to fuse selected restriction enzymes (here EcoRV) with an engineered Rop protein which forms a stable extended coiled coil [3]. The prepared protein-protein fusions will then be fluorescently labeled using appropriate fluorophores. The labeled constructs will be characterized extensively by biochemical and biophysical techniques, before they can be studied in their interaction with DNA using single molecule techniques.