Protamine progressively folds DNA before looping

In sperm, protamine proteins cause a rapid condensation of the DNA to almost crystalline packing levels. Understanding the physical mechanism for this dramatic process is an important limiting case in biophysics. In addition, protamine condensation of DNA may be useful in biomaterials research to assemble or fold DNA nanostructures. Here our goal is to understand the first step in the pathway, the folding of the DNA into a single loop by protamine. To answer this question, we image single molecules of DNA with bound protamine using an atomic force microscope (AFM). We also directly measure DNA folding dynamics in real time using tethered particle motion (TPM) assays. We expected our measurements to show a single folding event as the loop forms, but both measurements showed the presence of multiple, long-lived (100 s) intermediates. Structurally, these intermediates are half-loops or “c-shapes” that become more folded over as the protamine concentration increases. This data suggests a new model for DNA looping whereby small molecules coat the DNA and progressively fold it before forming a loop.