Measuring jumping during DNA target search

We are investigating the DNA target search mechanisms of type II restriction endonucleases. Here we present our work in developing a method to visualize jumping by DNA binding proteins as they search a DNA. We have previously shown that EcoRI translocates along DNA with a salt dependent diffusion coefficient. We have shown that at low salt, the protein primarily slides in contact with the DNA, whereas at higher salt, hopping increasingly contributes to the one dimensional diffusion. Whereas hopping is from one position to a nearby position in a linear DNA, jumping occurs in coiled DNA and allows a DNA binding protein to land on a distant segment of the DNA. Jumping is difficult to visualize due to the rapidly fluctuating coiled DNA. In our method, we will alternately stretch and coil a DNA using dual optical tweezers, while tracking a fluorescently labeled EcoRI. Visualizing large displacement during coiling phases will be evidence of jumping events. We have expressed and purified a cleavage deficient mutant that we are labeling with a quantum dot to allow us to track the movement of the endonuclease in real time. We intend to use this labeled mutant in our single molecule tracking experiments.