Date Available

8-1-2016

Year of Publication

2016

Document Type

Master's Thesis

Degree Name

Master of Science (MS)

College

Arts and Sciences

Department/School/Program

Chemistry

Advisor

Dr. Jason DeRouchey

Abstract

In sperm chromatin, DNA exists in a highly condensed state reaching a final volume roughly twenty times that of a somatic nucleus. For the vast majority (>90%) of sperm DNA in mammals, somatic-like histones are first replaced by transition proteins which in turn are replaced by arginine-rich protamines. This near crystalline organization of the DNA in mature sperm is thought crucial for both the transport and protection of genetic information since all DNA repair mechanisms are shut down. Recent studies show that increased DNA damage is linked to dysfunctions in replacing histones with protamines resulting in mispackaged DNA. This increased DNA damage correlates not only to infertility but also impacts normal embryonic development. This damage is currently poorly characterized, but is known to involve oxidative base damage by reactive oxygen species (ROS).

Using a variety of biophysical methods, the effect of DNA condensation by polycations on the on free radical access and DNA damage in the packaged state was investigated. In Chapter 2, gel electrophoresis was used to quantify the ability of free radicals to damage both unpackaged and packaged DNA. DNA condensed by polycations shows significantly reduced levels of indirect damage from exposure to free radicals. Combining previous work on packaging density, it is also shown that differences in the packaged state, even by a few Angstroms, can result in significantly different degrees of damage to the DNA. In Chapter 3, we investigate the effects of protamine concentration on the ability to condense and protect DNA. Insufficient protamination is known to be a potential source of protamine dysfunction in mammalian sperm chromatin. Using gel retardation assays and UV-Vis studies, we examined the ability for DNA to condense with protamine at varying nitrogen to phosphate (N:P) charge ratios. Initial results on damage as a function of N:P are also discussed. Future work will more quantitatively determine the interrelationship between DNA packaging densities and the resulting accessibility of DNA to reactive oxygen species (ROS).

Digital Object Identifier (DOI)

http://dx.doi.org/10.13023/ETD.2016.348

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