Doctor of Philosophy

dissertationOxidative damage to DNA, a factor in cancer, mutation, and aging, is attributed to reactive oxygen species (ROS). ROS are formed exogenously and endogenously and attack DNA showing a preference for reaction with 2-deoxyguanosine (dG) sites. In present work, dG was oxidized by HO. that was generated via iron-Fenton reaction or by X-ray radiolysis of water. Under aerobic conditions and in presence of reductant, the product 5-carboxamido-5-formamido-2-iminohydantoin (d2Ih) was the major lesion formed. The above prescribed conditions are mimic to cellular conditions. DNA polymerase and base excision repair processing of 2Ih lesion has been evaluated in this study. 2Ih bears a stereocenter in the base and was found as two diasteromers, (R)-2Ih and (S)-2Ih. Single nucleotide insertion opposite (R)-2Ih and (S)-2Ih in the template strand catalyzed by the DNA polymerases Klenow fragment exo?, DPO4, and Hemo KlenTaq demonstrate these lesions cause point mutations. Specifically, they promote thr

Computational studies of electronic circular dichroism spectra predict absolute configuration assignments for the guanine oxidation product 5-carboxamido-5-formamido-2-iminohydantoin

Journal ArticleOxidation of the guanine heterocycle by two electrons can yield the chiral product 5-carboxamido-5-formamido-2-iminohydantoin (2Ih). The 2Ih free base enantiomers were synthesized from 2'-deoxyguanosine oxidized with a Cu(II)/H2O2 oxidant system followed by hydrolysis of the N-glycosidic bond. These isomers were each studied by electronic circular dichroism spectroscopy for determination of their absolute configurations. Time-dependent density functional theory calculations of the expected spectra were completed in both the gas phase and with solvent modeling in order to interpret the experimental spectra and provide the absolute configuration assignments

Rates of Chemical Cleavage of DNA and RNA Oligomers Containing Guanine Oxidation Products

The nucleobase guanine in DNA (dG) and RNA (rG) has the lowest standard reduction potential of the bases, rendering it a major site of oxidative damage in these polymers. Mapping the sites at which oxidation occurs in an oligomer via chemical reagents utilizes hot piperidine for cleaving oxidized DNA and aniline (pH 4.5) for cleaving oxidized RNA. In the present studies, a series of time-dependent cleavages of DNA and RNA strands containing various guanine lesions were examined to determine the strand scission rate constants. The guanine base lesions 8-oxo-7,8-dihydroguanine (OG), spiroimino­dihydantoin (Sp), 5-guanidino­hydantoin (Gh), 2,2,4-triamino-2<i>H</i>-oxazol-5-one (Z), and 5-carboxamido-5-formamido-2-imino­hydantoin (2Ih) were evaluated in piperidine-treated DNA and aniline-treated RNA. These data identified wide variability in the chemical lability of the lesions studied in both DNA and RNA. Further, the rate constants for cleaving lesions in RNA were generally found to be significantly smaller than for lesions in DNA. The OG nucleotides were poorly cleaved in DNA and RNA; Sp nucleotides were slowly cleaved in DNA and did not cleave significantly in RNA; Gh and Z nucleotides cleaved in both DNA and RNA at intermediate rates; and 2Ih oligonucleotides cleaved relatively quickly in both DNA and RNA. The data are compared and contrasted with respect to future experimental design