Protamine Loops DNA in Multiple Steps

Abstract

DNA in sperm cells is incredibly compact. This compaction is mediated by the protein protamine, which loops DNA into a series of toroids. We are interested in the first step in the in vitro pathway which involves folding DNA into a loop. Previously, it was thought that DNA loop formation would occur in one-step with a mechanism similar to bacterial transcription factors. Bacterial transcription factors loop DNA by i) binding to one location, ii) waiting for a spontaneous fluctuation in the DNA to cause a loop, and iii) stabilizing the loop by binding to a second location. However, unlike bacterial transcription factors that bind one at a time, protamine is known to coat the DNA, binding every 10 bp or so. Thus, the mechanism of protamine might be different from bacterial transcription factors. To study the mechanism of loop formation, we used a tethered particle motion (TPM) assay to measure the dynamic, real-time looping of single DNA molecules. We observed that folding did not occur in a single step as predicted. Instead, the DNA folded multiple times into long-lived (∼100 s), reversible, folded states. We used an atomic force microscopy (AFM) assay to image the folded structures directly. We observed that the partially folded molecules were partially folded loops—c-shapes or s-shapes—that had a radius of curvature of ∼10 nm. Analysis of the contours of the molecules suggest that protamine is bending the DNA rather than increasing the flexibility of the DNA. Thus, our model is that protamine loops DNA in multiple steps, bending it into a curved structure as it binds. This novel pathway for loop formation may be used by other multivalent cations to loop or bend DNA. For more information, see published work in NAR 2020.