Year of Award

2007

Document Type

Thesis

Degree Type

Master of Science (MS)

Degree Name

Chemistry

Department or School/College

Department of Chemistry

Committee Chair

Kent Sugden

Keywords

DNA damage, DNA repair, histones, nucleosome

Abstract

Eukaryotic DNA is packaged in a condensed state with histone proteins. The minimal structural unit within packaged eukaryotic DNA is the nucleosome core particle (NCP). The NCP consists of a 146 bp DNA fragment wrapped around an octamer of histone core proteins. Nucleosome core particle formation induces DNA structural changes and reduced DNA accessibility providing a very different setting than that commonly modeled by in vitro studies. In vitro reconstituted NCP provide a controlled environment that more closely models eukaryotic DNA than studies using naked DNA. Reconstituted NCP studies of DNA damage have exhibited a spectrum of effects compared to naked DNA ranging from protective, enhanced, and no effect. The nature of the effect appears to be related to the type of oxidant, its sterics and interactions with the histone surface and altered DNA structure. While there is differences in efficiencies of oxidation of nucleobases throughout the nucleosome, nucleobase oxidation is still widespread within the genome. DNA repair processes that combat global DNA oxidation are crucial to cell survival. One major cellular repair mechanism that is employed to remove DNA damage is base excision repair (BER). The BER pathway involves the concerted activity of a small number of proteins which catalyze individual reactions in a chemical pathway that repairs single nucleotide lesions. In vivo, the majority of DNA is wrapped around histones and the repair machinery of BER has to work within or around the structure of the nucleosome and deal with a distorted DNA structure and reduced accessibility due to the presence of bulky histone proteins. To address the questions of DNA damage and repair in the nucleosome an in-vitro nucleosomal system was established by reconstituting purified histones and a 154 bp wrapping fragment from the Xenopus borealis 5S rRNA gene to form individual nucleosome core particles (NCP). The effect of nucleosome formation on chromium- mediated DNA damage and the efficiency of BER glycosylase cleavage of the lesion 8-oxoG were investigated. Base excision of 8-oxoG by Fpg and hOGG1 indicated that: i) the position of the lesion 8-oxoG in naked DNA can influence BER activity; ii) nucleosomal formation decreases the activity of these BER enzymes by as much as 2.5 fold with a rotational dependence exhibiting increased cleavage towards the more accessible lesion; iii) the rotational dependence for both Fpg and hOGG1 was almost identical, however hOGG1 showed better cleavage in the nucleosome setting relative to free DNA at earlier time points. An additional study was done to examine the potential of 8-oxoG lesions to mimic cytosine methylation effects with regard to the activity of a methyl-sensitive endonuclease. Using enzyme cleavage assays the effects of placing an 8-oxoG or methylated cytosine into the recognition sequence of a restriction endonuclease, NotI, were investigated. Results indicate identical inhibitory effects between 8-oxoG and cytosine methylation, hinting at a potential role of 8-oxoG in epigenetics.

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© Copyright 2007 James Joseph Covino