Date of Graduation
Master of Science in Chemistry
Chemistry and Biochemistry
DNA damage, such as single base lesions and mismatches, is highly prevalent within cells. If these DNA damage events are not repaired, they could lead to mutations and thus disease and cancer. Intricate repair mechanisms are in place to fix these damage events, one such being Base Excision Repair (BER) and associated enzyme: Thymine DNA Glycosylase (TDG). The first step of this repair process, recognition of the lesion by TDG, is not well understood. The following thesis presents results to better understand the fundamental biophysical question of how a DNA lesion within a mismatch context is recognized in a million fold excess of normal DNA bases. Part one of this research involves the investigation of an 8mer non-palindromic DNA sequence containing a U:G mismatch and how this mismatch alters the phosphodiester backbone of the DNA around the lesion site. Results showed that there was a stepwise difference in the free energy of the conformational equilibrium of the DNA backbone around the lesion site, following trends that have been shown for palindromic DNA sequences. Part two investigates attempts to optimize the expression and purification conditions for Thymine DNA Glycosylase. Part three involves analyzing how DNA sample conditions influence the backbone conformational equilibrium dynamics within a dodecamer palindromic sequence containing a T:G mismatch. Of particular interest, the free energy was impacted for the lesion sequence at the phosphate 3ʹ to the base-pairing partner in the presence of potassium and magnesium ions and impacted to a lesser extent at the phosphate 3ʹ to the lesion in the presence of potassium ion. For the DNA analysis of the phosphodiester conformational equilibrium dynamics, 1-D and 2-D Nuclear Magnetic Resonance (NMR) Spectroscopy was used.
DNA, Base Excision Repair, Thymine DNA Glycosylase, 1D and 2D solution Nuclear Magnetic Resonance Spectroscopy, BI/BII, dynamics, conformations, equilibrium, sample conditions
Biochemistry | Biochemistry, Biophysics, and Structural Biology | Biophysics | Chemistry | Physical Chemistry
© Autumn C. Pilarski
Pilarski, Autumn C., "Impact of Sample Conditions on DNA Phosphodiester Backbone BI/BII Conformational Equilibrium Dynamics" (2023). MSU Graduate Theses. 3885.