Biological Sciences, School of
Date of this Version
5-2016
Abstract
Nucleosomes and the secondary structure of DNA play an important role in many cellular processes that are contingent on DNA accessibility, such as replication, transcriptional regulation, and DNA repair. Recently, there is also growing evidence that the association of histones and DNA to form a nucleosome offers protection from DNA degradation. Specifically, that the inherent structural properties of DNA organization can help protect DNA from exogenous damage such as radiation. In this study, we sought to evaluate the effects of radiation on the positioning of histones and other proteins.
In this experiment, we optimized established protein protocols in Saccharomyces cerevisiae. Briefly, yeast cells were exposed to radiation, isolated, and then enzymatically treated with nucleases that digest regions of DNA that aren’t bound to a protein. Two enzymatic digestions were performed to determine the positioning of histone-bound DNA as nucleosomes and likewise, the positioning of all protein-bound DNA, histones and non-histones alike. The DNA was purified and then quantitative real-time PCR with multiple primer sets was used to analyze a particular region of DNA. By comparing enzymatic digestion amplicon recovery, we were able to determine the relative protection value for each ~100 bp region of DNA and evaluate whether it was protected from digestion or not.
We evaluated the effects of two, single doses of radiation on the positioning of proteins in yeast cells. This system was subsequently applied to evaluate eight doses of a higher amount of radiation. Four loci were evaluated and we found that the pattern of nucleosome positioning and other proteins was conserved regardless of radiation exposure. Lastly, we applied the system to evaluate three forensically important Short Tandem Repeat (STR) loci in human white blood cells. We also found that the pattern of positioning was conserved. Our work may suggest that nucleosomes and other proteins play an important role in protecting DNA, illustrated by the conservation of positioning regardless of radiation exposure. This work advocates for the importance of the intrinsic structural properties of DNA and the use of such information to aid in the selection of regions of DNA that are more likely to be protected in situations of degradation.
Advisors: Ashley M. Hall and Fred P. Baxendale
Comments
A THESIS Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Master of Applied Science, Major: Applied Science, Under the Supervision of Professors Ashley M. Hall and Fred P. Baxendale. Lincoln, Nebraska: May, 2016
Copyright 2016 Ashley E. Foster