Protamine folds DNA into flowers and loop stacks

DNA in sperm undergoes an extreme compaction to almost crystalline packing levels. To produce this dense packing, DNA is dramatically reorganized in minutes by protamine proteins. Protamines are positively charged proteins that coat negatively charged DNA and fold it into a series of toroids. The exact mechanism for forming these ∼50-kbp toroids is unknown. Our goal is to study toroid formation by starting at the “bottom” with the folding of short lengths of DNA that form loops and working “up” to more folded structures that occur on longer length scales. We previously measured the folding of 200–300 bp of DNA into a loop. Here, we look at folding of intermediate DNA lengths (L = 639–3003 bp) that are 2–10 loops long. We observe two folded structures besides loops that we hypothesize are early intermediates in the toroid formation pathway. At low protamine concentrations (∼0.2 μM), we see that the DNA folds into flowers (structures with multiple loops that are positioned ..., DNA construct preparation and protamine
DNA lengths of 639, 1170, and 3003 bp were amplified from Lambda DNA using standard PCR procedures (41,51). We used an LA Taq DNA polymerase (TaKaRa Bio RR002) and custom primers (Integrated DNA Technologies). Forward primers were tagged with biotin, and reverse primers were tagged with digoxigenin so that the final DNA product could bind to the streptavidin-coated particle and an anti-digoxigenin-coated slide, respectively. GC content for each construct was 52% (639 bp), 33% (1170 bp), and 44% (3003 bp). We have previously seen that loops are more likely to form in regions of lower GC content (52). Other studies (53) have seen that the presence of static curvature in the DNA created by phased tracts of poly(A) regions has led to loops within toroids that have reduced diameters.
After amplification, we checked the purity of our product using a gel purification procedure. We first performed gel electrophoresis using orange loadi..., # Data from: Protamine folds DNA into flowers and loop stacks

Dataset DOI: [10.5061/dryad.k6djh9wnf](https://doi.org/10.5061/dryad.k6djh9wnf)

## Description of the data and file structure

### Files and variables

#### File: McMillan_BiophysJ_2023_Library_of_Singlets.zip

**Description:** 

This library contains images (.jpg files) of single DNA molecules (singlets) taken with an AFM. Images are organized into folders by sample conditions, specifically the length of the DNA (217, 398, or 1023 nanometers) and the protamine concentration (0, 0.2, 0.6, 2.0 micromolar, etc.).

## Code/software

Any software that can open .jpg files can be used to view the images.

## Access information

Data was derived from the source: [10.1016/j.bpj.2023.10.003](https://doi.org/10.1016/j.bpj.2023.10.003), ,