UC Riverside

Modifications to deoxyribonucleic acids (DNA) may arise endogenously or be initiated by exogenous sources. In this dissertation, we focus on utilizing mass spectrometry (MS), liquid chromatography (LC), and density functional theory (DFT) methods to characterize DNA modifications induced by chemotherapeutic agents, and to assess the roles kinetics and thermodynamics play in the removal of oxidized pyrimidines by human thymine DNA glycosylase (hTDG).

In part I, we conducted an enzymatic digestion study coupled with detailed MS, tandem MS (MS/MS), and LC-MS/MS analyses of oligodeoxyribonucleotides (ODNs) containing cisplatin adducts (Chapter 2). We identified and characterized several digestion products for ODNs containing a 1,2-GpG, 1,2-ApG or 1,3-GpXpG intrastrand cross-link. These results built a solid foundation for the future LC-MS/MS quantification of these DNA lesions.

Additionally, we characterized the threshold energy of cross-link formation based upon the UVB irradiation of ODNs harboring a 5-halouracil or 5-halocytosine (Chapter 3). We found that the calculated threshold energies for 5-halocytosines were more exothermic than the corresponding 5-halouracils, and more exothermic for 5-iodopyrimidines than for the 5-bromopyrimidine counterparts, suggesting greater cross-link formation for the former pyrimidine derivatives. These calculation results were in excellent agreement with reported experimentally determined yields of the cross-link products and supported the mechanistic proposal that cross-link products form from UV-induced electron transfer followed by heterolytic cleavage of the C5-X bond.

In part II, we focused on the characterization of hTDG activity towards oxidized pyrimidine base modifications formed at methyl-CpG site. We used DFT methods to assess the kinetic and thermodynamic parameters for the cleavage of the N-glycosidic bond of 5-methyl-2'-deoxycytidine (mdC) and its, (1) oxidation products as it pertains to a newly proposed mechanism for active cytosine demethylation (Chapter 4); and (2) oxidation and subsequent deamination products [i.e., the 2'-deoxyuridine (dU) derivatives] (Chapter 5). Our studies suggest that the inherent chemistry associated with the nucleophilic cleavage of the N-glycosidic bond constitutes a major factor contributing to the selectivity of hTDG towards 5-substituted dC and dU derivatives. These findings provided novel chemical insights into the role TDG may play in maintaining the epigenetic integrity of the CpG site.