Characterizing the Mechanism of DNA Loop Formation by Protamine

DNA compaction in sperm cells by the protein protamine represents an extreme case of polymer folding, approaching crystalline packing of the molecule. This high compaction is necessary for creating viable sperm, yet the physical mechanism of folding is unknown. In this study, we investigate how protamine folds DNA into a loop. One hypothesis is loop formation occurs through entropic folding where protamine binds and neutralizes the negative charge of the DNA backbone, increasing flexibility and allowing the molecule to fold into a loop. Another hypothesis is loop formation occurs as each protamine binds and bends DNA, changing its enthalpy. Using single-molecule force spectroscopy, we measure force-extension curves and fit them with a worm-like chain model. This model obtains the entropic and enthalpic portions of the folding, allowing us to differentiate between our two hypotheses. Specifically, we pull on DNA tethers biochemically attached on one end to a glass slide and on the other to a polystyrene bead. We then flow protamine over the tethers and measure the force-extension curve. We use a centrifuge force microscope to apply force and to image the tethers to track their extension. Here we report progress in building the instrument and measuring force-extension curves.