CAREER: Linking Structure, Stability and Protection in Protamine Packaged DNA

Overview: The highly condensed, liquid crystalline organization of DNA in mature sperm is thought crucial for the protection of genetic information in the absence of DNA repair. Despite intense research, the biophysical mechanisms underlying tight packaging of DNA remain poorly understood especially at the molecular level. Spermiogenesis is a unique multi-step process resulting ultimately in the replacement of histones by protamines in sperm nuclei to a final volume roughly 1/20th of that of a somatic nucleus. Protamines are short, 50-110 residue, highly basic proteins that account for the vast majority (>90%) of DNA packaging in sperm chromatin and contain 50-80% arginine, ~10% cysteines used to form stabilizing disulfide bridges, several serine and threonine phosphorylation sites, and multiple histidine and cysteine pairs to coordinate zinc ions. Protamines are initially phosphorylated and only subsequently dephosphorylated upon completion of successful sperm chromatin remodeling. The unique use of phosphorylation sites, disulfide bonds, and zinc ions makes sperm chromatin a uniquely rich system to investigate the physical chemistry and structure of DNA in the compacted state. We propose the following hypotheses (i) Protamine chemistry and dysfunctions, including incomplete protamine dephosphorylation and/or insufficient protamination are a major source of defective sperm chromatin remodeling and (ii) mispackaging renders DNA more accessible to chemical agents that contribute significantly to DNA damage. Intellectual Merit: The goal of this CAREER proposal is to develop basic understanding and predictive capability of how cation chemistry influences fundamental DNA-DNA interactions in highly packaged states using sperm chromatin as a model system. To achieve our goals, two specific aims are proposed: (1) determine the mechanisms by which nature utilizes protamines to package DNA in sperm chromatin and (2) relate defects in chromatin packaging structure to decreased stability due to higher accessibility to chemical agents. A variety of structural, biophysical and biochemical methods will be employed to evaluate protamine-DNA structure and stability to free radicals. Direct measurement of interhelical forces in condensed DNA arrays with native and modified protamines will give a better understanding of the physical interactions underlying assembly of charged molecules in vitro and in vivo. Mass spectrometry, and other methods, will allow us to link packaging density to nucleic acid stability for protamine packaged DNA. Protamines have several novel aspects that can carry over to other synthetic systems used for DNA compaction. The outcomes from this research are not only expected to make significant and fundamental contributions to our understanding of DNA compaction in sperm chromatin but to also advance our understanding of intermolecular forces between charged surfaces which will inform the rational design of future agents for DNA delivery and biosensors. Broader Impacts: DNA self-assembly is a critical testing ground for understanding the physics of interactions between charged molecules. The proposed research spans the disciplines of molecular biology, biochemistry and analytical chemistry, to answer fundamental questions in molecular biophysics. Its multidisciplinary nature will create unique training opportunities for undergraduate and PhD researchers. Training and dissemination of research will be provided by publication in scientific journals and student participation in national meetings. The educational activities of the proposed work also include stronger chemical and biophysical education across several levels. Community outreach will be provided through the creation of massive open online courses (MOOCs) designed to bridge the gap in academic credentials of incoming high school teachers by allowing access to inexpensive or free instruction and course material. Teaching modules developed in the MOOCs will be integrated into various undergraduate and graduate courses at Kentucky. Summer workshops will be created for involvement of high school teachers to learn about and actively participate in refining and assessing the proposed MOOC courses to best meet the needs of the teachers and their students nationwide. Additional educational opportunities will be fostered by the continued involvement of the PI in the UKs Partnership Institute for Math and Science Education Reform (PIMSER) program for regional middle school students, Hope Hill Youth services and two NSF funded REU Summer Programs at Kentucky as well as participation at state government events.