Protamine Loops DNA in Multiple Steps

Protamine proteins dramatically condense DNA in sperm to almost crystalline packing levels. Here, we measure the first step in the in vitro pathway, the folding of DNA into a single loop. Current models for DNA loop formation are one-step, all-or-nothing models with a looped state and an unlooped state. However, when we use a Tethered Particle Motion (TPM) assay to measure the dynamic, real-time looping of DNA by protamine, we observe the presence of multiple folded states that are long-lived (similar to 100 s) and reversible. In addition, we measure folding on DNA molecules that are too short to form loops. This suggests that protamine is using a multi-step process to loop the DNA rather than a one-step process. To visualize the DNA structures, we used an Atomic Force Microscopy (AFM) assay. We see that some folded DNA molecules are loops with a similar to 10-nm radius and some of the folded molecules are partial loops-cshapes or s-shapes-that have a radius of curvature of similar to 10 nm. Further analysis of these structures suggest that protamine is bending the DNA to achieve this curvature rather than increasing the flexibility of the DNA. We therefore conclude that protamine loops DNA in multiple steps, bending it into a loop.