Oxidative DNA damage is a contributor to a wide range of health problems including cancer, aging and neurological disorders. 8-Oxo-7,8-dihydroguanosine (OG) is regarded as perhaps the most critical lesion resulting from oxidative DNA damage due to its capability to mispair with A and, if unrepaired, to lead to a G-*T transversion mutation. Recent studies in this laboratory and others have shown that OG is an unstable intermediate under a variety of oxidative conditions. The products of one-electron oxidation of OG have now been established in this lab as a spiroiminodihydantoin (Sp) derivative in ss DNA and in nucleosides, while the major product in duplex DNA is a mixture of two isomeric species, a guanidinohydantoin (Gh) and an iminoallantoin (Ia) analog. These lesions are also formed directly by oxidation of G using singlet oxygen and peroxyl radicals. The central hypothesis of this project is that the activity of these lesions (Sp and Gh/ Ia) with polymerases and DNA repair enzymes warrants further study because of (a) the frequency of formation of OG under conditions of oxidative stress, (b) the high reactivity of OG toward further oxidation compared to the normal DNA bases, (c) the ability of OG to act as a hot spot for further oxidative damage via long-range electron transfer in duplex DNA, and (d) the recent finding that these lesions are primary oxidation products of G.
The specific aims of this project are (1) to study the reaction conditions that lead to Sp, Gh and Ia lesions in vitro by comparison of one-electron oxidants to other oxidants such as singlet oxygen, superoxide, and hydroxyl radical, (2) to develop synthetic methods for incorporation of pure, well-characterized lesions into oligonucleotides, (3) to determine the structural effects of Sp and Gh/Ia on duplex oligomers through an analysis of (a) duplex stability and (b) NMR, (4) to examine the polymerase processing of DNA oligomers containing these lesions using a variety of DNA and RNA polymerases and (5) to investigate the DNA repair activity of these lesions with Fpg, yOggi, yOgg2, hOggi and MutT. The methodology will rely heavily on synthetic oligomers containing the various lesions, their structural analysis by LC-MS and by 2D-NMR, in vitro enzyme kinetic analysis and in vivo studies in E. coli. Collaborative studies will include investigations of Sp and Gh/Ia in in vivo mutagenesis studies.
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