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Master of Science (MS)
Smirnov, Serge L.
Spiegel, P. Clint
Antos, John M.
Most types of chemical modifications of DNA bases are endogenous processes which are sensitive to the intracellular conditions. For example, the enzymatically catalyzed methylation of canonical cytosine to 5-methylcytosine (5metC) is a key form of epigenetic regulation of gene expression patterns. This modification is catalyzed and controlled by DNA methyltransferases (DNMTs). Perturbed rates of enzymatic DNA methylation leads to hyper- or hypo-methylation, both of which are a common initiating step in several forms of cancer. In addition to epigenetics, DNA bases can be chemically altered, or damaged, in response to reactive oxygen species (ROS). The oxidation of DNA by ROS can lead to formation of various types of adducts, with 8-oxoguanine (oxoG) being one of the most prolific and toxic. oxoG is a deleterious modification which has been linked to cancer and neurological disorders. The first step in oxoG damage repair, oxoG glycosylase (hOGG1) recognizes and excises the oxidized base. Oxidation of guanine and methylation of cytosine can occur simultaneously within CpG sites. Moreover, in these sites the both enzymology of oxoG repair is compromised by the adjacent 5metC, and the enzymatic methylation of cytosine is altered by oxoG. This manuscript describes the structural study of the DNA substrates where oxoG and 5metC modifications are clustered in a CpG site to aid in the understanding of the enzymatic effects of such clustering. The NMR solution structure is shown of six of these proposed duplex DNA samples, two with a single oxidation, two with a single methylation, one sample with a fully methylated CpG site and a modified sample with both oxoG and 5metC occurring on opposing strands in one single base pair. No global structural changes are reported amongst these structures, with all of these structures featuring elements of right-handed A/B DNA. One local structural change was observed in all samples with oxoG, namely that oxoG causes the BII backbone conformation 3' of the modification site. This BII backbone conformation may be the link between these modifications and lower enzymatic activity, as both hOGG1 and DNMT1 make direct contact with the 3' backbone following an oxoG.
DNA damage, DNA repair, Active oxygen in the body, Carcinogenesis, Genetic toxicology
Western Washington University
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Hoppins, Joanna, "Comprehensive Solution Structure Analysis of DNA Duplexes Containing Chemical Base Alterations" (2016). WWU Graduate School Collection. 520.