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Master of Science (MS)
Smirnov, Serge L.
Spiegel, P. Clint
Anthony-Cahill, Spencer J.
All known living organisms use DNA to store genetic templates used for development, proper function and reproduction. The structural integrity of DNA is therefore of extreme importance and cellular machinery continuously regulates our DNA either through addition of covalent molecules to regulate the transcription of genes or the removal of DNA lesions propagating from the exposure to reactive molecules. One of the most common DNA lesions, 8-oxoguanine (8OG), is a prominent, pro-mutagenic DNA adduct present at a baseline level from consistent generation of reactive oxygen species through oxidative metabolism or at greater concentrations through exposure to ionizing radiation and other toxins. Its mutagenic potential is attributed to its ability in the syn- conformation, to mimic thymine during DNA replication, resulting in a mispair with adenine. In contrast, 5-methylcytosine (5MC), occurs from the covalent addition of a methyl group to a cytosine base by a DNA methyltransferase. 5MC acts as an epigenetic gene regulator, often found densely packed within CpG islands upstream of transcriptionally inactive genes. It can be estimated that each human diploid cell contains hundreds of CpG dinucleotides undergoing active methylation while also harboring 8OG. Previous results obtained by Kasymov et al, showed reduced endonuclease activity by hOGG1 for substrates containing 5MC adjacent and cross strand from 8OG. In addition, the work presented by Maltseva et al, conveyed that the enzymatic methylation rates by maintenance DNA methyltransferases were severely impacted when 8OG is adjacent to the methylation target. These results prompted us to investigate the clustering of these two modifications in greater detail.
We present the results of solution NMR structure determination, thermodynamic stability analysis and molecular dynamics simulations on the DNA sequence 5’-d(CGCGAATTCGCG)-3’ with clustered 5MC and 8OG in CpG dinucleotides. NMR spectroscopy and restrained molecular dynamics were used to refine the structure of 11 DNA duplexes containing different methylation and oxidation patterns. The results reveal that 8OG induces local unwinding 5’ to itself and 31P chemical shifts indicate an increase in the BII phosphate backbone conformation 3’ relative to 8OG. Melting temperatures of the duplexes was shown to decrease with the addition of 8OG in all contexts. Surprisingly, the addition of 5MC in two separate instances led to lower Tm values of already oxidized DNA samples. 1D-1H NMR linewidths indicate 8OG increases the base dynamics while incorporation of 5MC leads to a stabilizing effect. Our results indicate that addition of 8OG to a fully-methylated CpG induces a sequence dependent stabilizing effect. Molecular dynamics trajectories were analyzed for BI/BII phosphate conformation populations, conformational flexibility and local dynamics. Comparison of helical geometries and backbone angles indicated that our MD simulations accurately and reliably reproduced our NMR structures within one standard deviation. Principal component analysis was carried out to highlight the most dominant modes of motion for CpG sites with clustered 5MC and 8OG. Particularly, we report significant differences in concerted atomic displacements, with the 8OG:5MC base pair displaying the greatest dynamic effects.
Western Washington University
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Gruber, David R., "Clustering of Epigenetic Methylation and Oxidative Damage: Effects on Duplex DNA Solution Structure, Thermodynamic Stability and Local Dynamics" (2017). WWU Masters Thesis Collection. 611.