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 (∼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 ∼10-nm radius, and some of the folded molecules are partial loops—c-shapes or s-shapes—that have a radius of curvature of ∼10 nm. Further analysis of th..., , # Data from: Protamine loops DNA in multiple steps

Dataset DOI: [10.5061/dryad.ngf1vhj8d](https://doi.org/10.5061/dryad.ngf1vhj8d)

## Description of the data and file structure

### AFM assay data collection and analysis

We used a Dimension 3000 AFM (Digital Instruments) with a Nanoscope IIIa controller to image samples and PPP-XYNCSTR-model cantilevers (NanoSensors, resonant frequency = 150 kHz, force constant = 7.4 N/m, length = 150 μm, tip radius < 7 nm) ([Supplementary Figure S1A](https://oup.silverchair-cdn.com/oup/backfile/Content_public/Journal/nar/48/11/10.1093_nar_gkaa365/1/gkaa365_supplemental_file.docx?Expires=1775490687&Signature=VrDDQhsaDp-XrIgrCbawz809XCsrX7poqguk02ho2AsHulIOH2dOxqa6BCNosf1DJ07nphFvVnM6STsuUNe2SkyHR~XOnhGN1giud1kc~9AFjopbrhc8OfkVYePqXFz~idlYw~cx5E5-r9C40Zn~kvJJ2Wawxi~jzLcsvpUvwnF9yGve8jO7P2YzcSDXy0xlR6nEM9JufoLEEilLAMB5w~1A9eyvRKDg3Gnqz9-wOl~NaGuFTbmNvUkB6YrmVSW7EuOAtHguPrfejAOx2tR~1zHeGhnVR8dMMVgHQA9xu6Y1UW8YUw9jHeU4HSziZLmsUB1UQqI48vER1ne8utmcIg__&Key..., ,