Optimizing Tethered Particle Motion to Measure DNA Compaction by Protamine

Abstract

During mammalian spermiogenesis, there is a remarkable change in the nuclear morphology whereby the chromatin is completely remodeled. Specifically, the histone proteins that wrap the DNA into nucleosomes are removed, and the DNA is coated with a series of small (∼100-amino-acid), arginine-rich protamines that dramatically compact the DNA into a series of toroids. This replacement and compaction 1) reduces the head size of the sperm to enhance their hydrodynamicity, 2) decreases the likelihood of DNA damage, and 3) removes the epigenetic markers passed on through histone modifications. Here, we directly observe the steps of toroid formation and the underlying mechanics. We use a single molecule biophysics assay where we tether a particle (1-µm-diameter bead) to the DNA and to the sample surface. During DNA folding, the end-to-end length of the DNA decreases, decreasing the motion of the particle. This tethered particle motion (TPM) assay is useful because we do not apply any external forces, allowing us to measure reversible folding events. Previously, we saw ∼200 nm changes in length that suggest that the toroid forms by successive loops. Here, we optimized the assay to enhance efficiency and resolution by improving bead monodispersion, protein binding to the surface, and DNA purification.