MANIPULATION OF DNA TOPOLOGY USING AN ARTIFICIAL DNA-LOOPING PROTEIN

Abstract

DNA loop formation, mediated by protein binding, plays a broad range of roles in cellular function from gene regulation to genome compaction. While DNA flexibility has been well investigated, there has been controversy in assessing the flexibility of very small loops. We have engineered a pair of artificial coiled-coil DNA looping proteins (LZD73 and LZD87), with minimal inherent flexibility, to better understand the nature of DNA behavior in loops of less than 460 bp. Ring closure experiments (DNA cyclization) were used to observe induced topological changes in DNA upon binding to and looping around the engineered proteins. The length of DNA required to form a loop in our artificially rigid system was found to be substantially longer than loops formed with natural proteins in vivo. This suggests the inherent flexibility of natural looping proteins plays a substantial role in stabilizing small loop formation. Additionally, by incrementally varying the binding site separation between 435 bp and 458 bp, it was observed that the LZD proteins could predictably manipulate the DNA topology. At the lengths evaluated, the distribution of topological products correlates to the helical repeat of the double helix (10.5 bp). The dependence on binding site periodicity is an unequivocal demonstration of DNA looping and represents the first application of a rigid artificial protein in this capacity. By constructing these DNA looping proteins, we have created a platform for addressing DNA flexibility in regards to DNA looping. Future applications for this technology include a vigorous study of the lower limits of DNA length during loop formation and the use of these proteins in assembling protein:DNA nanostructures.