The impact of DNA twisting and bending rigidity on protein–induced looping dynamics

Protein–induced DNA looping is a key regulatory mechanism involved in important processes such as transcription, gene regulation and replication. The relationship between the induced loop topology and DNA–protein dynamics is essential for understanding these processes. Here, we use tethered–particle motion (TPM) to examine the impact of bending and twisting rigidity of protein–induced DNA looping using the restriction endonuclease FokI as a test system. To cleave DNA efficiently, FokI bridges two copies of an asymmetric target, which can be arranged in either inverted or directly–repeated orientations. Using a combination of FRET and TPM, we show that in either arrangement, FokI generates a single looped species with the sites aligned in a parallel synapse. Furthermore, we show that both site separation and orientation have a profound influence on the dynamics of the looped DNA–protein structures. Surprisingly, the presence of a nick within the loop does not affect the observed rigidity of the DNA. In contrast, introducing a 4 nucleotide gap fully relaxes all of the torque present in the system but does not necessarily enhance loop stability. FokI therefore employs torque to enhance its binding by acting as a torsional catch bond.