Investigation of GapR protein-mediated DNA softening and supercoiling using magnetic tweezers

DNA topology plays a crucial role in the cell cycle. One of the most common forms of DNA topology is supercoiling. One approach to determine DNA supercoiling is chromatin immunoprecipitation (ChIP) sequencing of GapR protein, a bacterial protein that preferentially binds overtwisted DNA. Through recent studies, in vivo, GapR was thought to remove positive supercoiling caused by type II topoisomerases during the process of replication and transcription. However, the binding and deformation properties of GapR remain incompletely characterized. Here, we use single-molecule magnetic tweezers to study the binding of GapR to DNA, and its effect on DNA stiffness and twisting. Using the magnetic tweezer on 10kb linearized pFOS1 DNA, we measured the effect of varying GapR concentration on the extension of pFOS1 at different superhelical densities and forces. We found that the GapR protein decreases persistence length and unwinds pFOS1 DNA, shifting the extension versus linking number “hat” curve. We also find that GapR protein supercoils the pFOS1 DNA in a cooperative manner described by the titration curve.