In most cells, DNA is wrapped around histones. However, when sperm cells are formed, these histones are replaced by small, positively charged molecules called protamines. Understanding the mechanism of this histone-to-protamine replacement pathway is important in the field of male infertility. Specifically, if the ratio of protamines (P1 and P2) in humans is incorrect, this can lead to abnormal replacement and lower sperm motility. Our hypothesis is that the direct histone-to-protamine replacement pathway occurs through an active displacement method in which protamines bind directly to the DNA and actively displace histones. In order to investigate this hypothesis, we would like to use atomic force microscopy (AFM) to visualize the folded states. The first step in the assay involves successfully producing enough purified DNA of our desired length. Next, we bind histones to the DNA using nucleosome assembly protein and run a partial digestion assay to verify binding. Finally, we visualize the histone bound DNA in the AFM with and without protamine. Previously, we were unable to construct full nucleosomes and only measured replacement of partial nucleosomes with 2–6 histones. Here, we will present work toward making full nucleosomes and visualizing the DNA at different folding states to eventually decode the role of protamines in male infertility.
Craig et al. (Sun,) studied this question.