In contrast to the damage caused by far-UV, the damaging effects of UVA (320-400 nm) in living cells are not well understood. The damage caused by UVA irradiation is largely oxygen-dependent, suggesting UVA-mediated DNA damage involves reactive oxygen species produced through the action of an endogenous photosensitizer. Previous studies examining cellular responses to UVA irradiation in E. coli have been hindered by the fact that, at sublethal fluences, wild-type cells undergo a transient inhibition of cell growth termed a "growth delay". This effect is absent in nuvA⁻ strains, thereby facilitating the study of DNA repair factors required for the repair of UVA-mediated damage. Formamidopyrimidine (Fapy) glycosylase (encoded by fpg) and the UvrABC excinuclease are both capable of excising oxidatively damaged DNA bases. An fpg::kan mutation was placed into isogenic uvrA⁺ and uvrA⁻ strains of E. coli to evaluate the relative importance of these repair enzymes in the recovery from UVA-induced stress. In a nuvA⁻ background, the survival of fpg⁻ mutants exposed to UVA was significantly reduced relative to isogenic fpg⁺ control strains. This effect was enhanced in the absence of the UvrABC excinuclease, suggesting a role for both of these enzymes in repairing UVA-generated lesions. Survival of isogenic nuvA⁺ repair-deficient strains was significantly lower than nuvA⁻ strains, suggesting a role for the modified base 4-thiouridine in UVA-mediated lethality. An in vitro plasmid DNA irradiation assay in the presence and absence of 4-thiouridine was used to examine this possibility. When irradiated DNA was subsequently used to transform the fpg⁻ and uvrA⁻ mutant strains, no increase in DNA damage (as measured by a decrease in transformational efficiency) in the presence of 4-thiouridine was observed, suggesting that when present in solution this base does not play a photosensitizing role in UVA-mediated lethality. / Thesis / Master of Science (MSc)

A sensitive and specific assay aimed at measuring the oxidized nucleic acids, 8-oxoguanine (8-oxoGua), fapy-guanine (Fapy-Gua), 8-oxoguanosine (8-oxoGuo), 8-oxo-2′-deoxyguanosine (8-oxodG) has been developed by coupling reversed phase liquid chromatography (HPLC) with electrospray tandem mass spectrometry detection (MS/MS) and isotope dilution. The HPLC-MS/MS approach with multiple reaction monitoring (MRM) allowed for the sensitive determination of 8-oxoGua, Fapy-Gua, 8-oxoGuo, and 8-oxodG in human urine samples. There is no sample preparation needed except for the addition of buffer and 13C- and 15N-labeled internal standards to the urine prior to sample injection into the HPLC-MS/MS system. This method was tested in urine samples from non-smokers, smokers, non-smokers with chronic kidney disease (CKD) and smokers with CKD, to assess the level of oxidative damage to nucleic acids. Markers of both RNA and DNA damage were significantly increased in the smokers with and without CKD compared to their respective control subjects. These findings suggest that a highly specific and sensitive analytical method such as isotope dilution HPLC-MS/MS may represent a valuable tool for the measurement of oxidative stress in human subjects.