Author ORCID Identifier

https://orcid.org/0000-0001-9801-6972

Date Available

8-13-2027

Year of Publication

2025

Document Type

Doctoral Dissertation

Degree Name

Doctor of Philosophy (PhD)

College

Arts and Sciences

Department/School/Program

Chemistry

Faculty

Jason DeRouchey

Abstract

In nature, DNA compaction is driven by cationic proteins like histones in somatic nuclei and arginine-rich protamines in sperm chromatin. Sperm nuclei are significantly more compact than somatic nuclei, with a volume around 1/20th that of somatic nuclei. In-vitro studies show a need for a cationic charge of +3 or higher to initiate DNA condensation; however, divalent cations like zinc cannot condense DNA independently. Protamines, the key players in sperm chromatin compaction, exhibit variability across species. For instance, human protamine P2 contains cysteine residues believed to interact with zinc. Intriguingly, protamines in other animals, including fish, birds, reptiles, and most marsupials, lack cysteine residues. Additionally, piscine protamines, such as those found in salmon, also lack histidine residues. Given the absence of cysteine or histidine residues in salmon protamine to coordinate with zinc, it was initially anticipated that salmon nuclei would lack this metal. However, analysis of sperm seminal fluid from two species of the Salmonidae family has revealed the presence of zinc along with calcium and magnesium. However, there are no studies to date regarding the structural role of these divalent metals in cysteine/histidine-devoid protamine-mediated sperm DNA packaging.

In this dissertation, my research focuses on how the divalent cations affect the in-vitro and in-vivo condensed DNA. Specifically, in Chapter 2, we studied zinc-induced tightening of DNA packaging in piscine sperm chromatin. Our small-angle X-ray scattering (SAXS) analysis reveals that adding low levels of zinc to salmon sperm nuclei leads to tighter DNA packing, a phenomenon also observed with other divalent cations like transition metals, alkaline earth metals, and alkylamines. Higher divalent concentrations exhibit a crossover effect, reducing DNA packing density at high salt levels. Inductively coupled plasma mass spectrometry (ICP-MS) quantification of naturally occurring zinc and other metals in salmon sperm nuclei suggests that protamine and divalent metals may be essential for optimal DNA stabilization in sperm chromatin. In Chapter 3, we investigated the selective influence of divalent cations on DNA packaging in polyplex. Our experiments show that zinc-induced tight packaging is universal in condensed DNA with positively charged peptides, such as arginine found in sperm protamine and lysine predominantly present in somatic histones. However, zinc doesn’t affect DNA packaging condensed by polyamine-like spermine, suggesting a preference for the -C terminal. Nonetheless, zinc shows tightened packing of DNA condensed by peptide with amidated C-terminal residues. Interestingly, unlike zinc, other divalent metals such as Mg2+ do not promote tight packing in peptide-DNA polyplexes. Circular dichroism experiments confirm that only zinc interacts with positively charged peptides, while other divalent metals do not exhibit this interaction. In Chapter 4, we studied the synergistic effects of divalent cations in DNA condensation by polycations. We investigated how the divalent cations affect DNA condensation by polycations. Our study shows that divalent cations exhibit cooperative attractive interactions, particularly zinc, which induces more significant condensation compared to other metals like magnesium. Investigations with small peptides suggest that zinc might add charges to the peptide chain to condense DNA, while other metals may merely neutralize phosphate charges and/or bind to bases. The findings outlined in this dissertation shed more insight into understanding how different divalent metals modulate DNA packaging dynamics in conjunction with polycation interaction.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2025.435

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Available for download on Friday, August 13, 2027

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