Mass Spectrometric Investigation on the Formation and Repair of DNA Modifications
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Mass Spectrometric Investigation on the Formation and Repair of DNA Modifications


The advancement of mass spectrometry (MS) facilitates sensitive and unambiguous analysis of DNA modifications. There are two subjects in this area of investigation: 1) discovery of novel DNA modifications; 2) revelation of repair mechanisms of DNA modifications. In this dissertation, we developed LC-MS/MS methods to quantify a number of DNA modifications in mammalian cells and to assess their repair. In Chapter 2, we employed nLC-nESI-MS/MS coupled with isotope-dilution method that simultaneously quantifies three pyridylhydroxybutyl (PHB) adducts induced by tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). Among them, O4-[4-(3-pyridyl)-4-hydroxylbut-1-yl]-thymidine (O4-PHBdT) was first discovered in mammalian genome upon NNAL exposure. The newly discovered O4-PHBdT, as well as O2-PHBdT and O6-PHBdG, was found to display dose-dependent formation both in vivo and in vitro. In addition, NER was shown to be involved in the removal of O2-PHBdT and O4-PHBdT, while direct reversal suicide protein MGMT may counteract the formation of O6-PHBdG and O4-PHBdT. In Chapter 3, we employed metabolic labeling to selectively incorporate N2-methyl-dG (N2-MedG) and N2-n-butyl-dG (N2-nBudG) into genomic DNA of cultured cells, and investigated how the levels of the two lesions in cellular DNA are modulated by different DNA repair factors. It was found that NER exerts moderate effects on the removal of N2-MedG and N2-nBudG from genomic DNA. Translesion synthesis (TLS) polymerases κ and η may contribute to the incorporation of N2-alkyl-dG into genomic DNA. Pol κ was found to be involved in the repair of both N2-MedG and N2-nBudG; while Pol η was responsible for the repair of less bulky N2-MedG, but not N2-nBudG. In addition, loss of ALKBH3 resulted in higher frequencies of N2-MedG and N2-nBudG incorporation into genomic DNA, suggesting a role of oxidative dealkylation in the reversal of these lesions. In Chapter 4, we extended the metabolic labeling approach to accommodate genomic incorporation of 2-amino-2'-deoxyadenosine (dZ) and investigated its plausible repair mechanism. Z is a naturally occurring non-canonical nucleobase that has been found in bacteriophages in substitution of adenine (A). It was observed for the first time that dZ could be incorporated into genomic DNA in mammalian cells, and TLS polymerases ι and REV1 may contribute to its incorporation. In addition, transcription-coupled NER (TC-NER), but not global genome NER (GG-NER), participates in the removal of dZ from human genome.

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