THE ROLE OF CHARGE ON DNA PACKAGING AND INTEGRITY WITHIN RECONSTITUTED PEPTIDE-DNA ASSEMBLIES
In nature, DNA exists primarily in a highly compacted form. The compaction of DNA in vivo is mediated by cationic proteins; histone in somatic nuclei and arginine-rich peptides called protamines in sperm chromatin. The packaging in the sperm nucleus is significantly higher than somatic nuclei resulting in a final volume roughly 1/20th that of a somatic nucleus. This tight packaging results in a near crystalline packaging of the DNA helices. While the dense packaging of DNA in sperm nuclei is considered essential for both efficient genetic delivery as well as DNA protection against damage by mutagens and oxidative species, the degree to how the DNA packaging directly relates to DNA protection in the condensed state remains poorly understood. It is known that protamines are initially phosphorylated by kinases and later, after protamine complexation with DNA, extensively dephosphorylated in the late stages of spermatogenesis before spermatozoa enter the epididymis. The observance of phosphorylated protamines in mature sperm is thought to arise due to partial or complete failure of protamine dephosphorylation during sperm maturation. It is thought that alteration of the phosphorylation/dephosphorylation process of protamines likely impacts DNA packaging in sperm cells negatively and thus, allows greater access to radical damaging species to accumulate and degrade sperm DNA.
In this dissertation, my research focuses on the role of polycation length, or equivalently charge, and phosphorylation on DNA packaging and DNA integrity within reconstituted sperm chromatin-like structures. Specifically, in chapter 2, we studied how different polycation chemistries and lengths altered the packaging of DNA and susceptibility to free radical damage within condensed DNA nanoparticles. This work reveals that the polycation dynamics, not simply the resulting DNA packaging density, plays a significant role in the resulting DNA integrity to insult by free radicals within the condensed DNA phase. In chapter 3, we investigated the impact of phosphorylation as a posttranslational modification on sperm chromatin structure. We systematically investigated on how protein chemistry, specifically the incorporation of either neutral or negatively charged phosphorylated amino acids into protamine mimic peptides, can be used to control the DNA-binding capacity, resulting DNA packaging density, and the resulting free radical-induced damage within DNA condensates. These studies show that phosphorylated amino acid residues are particularly effective in reducing DNA packaging density resulting in significantly higher accumulation of Fenton chemistry mediated-radical damage to the DNA. We also show that while radical scavengers ameliorate DNA damage, this is attenuated in DNA packaged with a phosphorylated oligoarginine peptide. Testes and spermatogonia are known to be more sensitive to ionizing radiation than other types of cells present in the body likely due to a lack of antioxidant enzymes and DNA repair. Radiation exposure can damage DNA both by direct ionization as well as generation of hydroxyl radicals that damage DNA. In Chapter 4, we directly studied the impact of X-ray irradiation on DNA damage within reconstituted peptide-DNA assemblies. Using our protamine mimic oligopeptides, we show the incorporation of negatively charged moieties, such as residual phosphorylation of protamines within sperm chromatin, results in significantly higher rates of damage to ionizing radiation. Using bacterial transformation assay, we also show a 3-fold decrease in viability of irradiated plasmid extracted from peptide-DNA assemblies containing a phosphoserine residue. The findings in this dissertation shed more insight into understanding how polycation chemistry modulate DNA packaging and accessibility to free radical damage.