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.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Chemical Pathology Study Section (CPA)
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Okano, Paul
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University of Utah
Schools of Arts and Sciences
Salt Lake City
United States
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Rogers, R Aaron; Fleming, Aaron M; Burrows, Cynthia J (2018) Unusual Isothermal Hysteresis in DNA i-Motif pH Transitions: A Study of the RAD17 Promoter Sequence. Biophys J 114:1804-1815
Fleming, Aaron M; Zhu, Judy; Ding, Yun et al. (2018) Human DNA Repair Genes Possess Potential G-Quadruplex Sequences in Their Promoters and 5'-Untranslated Regions. Biochemistry 57:991-1002
Omaga, Carla A; Fleming, Aaron M; Burrows, Cynthia J (2018) The Fifth Domain in the G-Quadruplex-Forming Sequence of the Human NEIL3 Promoter Locks DNA Folding in Response to Oxidative Damage. Biochemistry 57:2958-2970
Zhu, Judy; Fleming, Aaron M; Burrows, Cynthia J (2018) The RAD17 Promoter Sequence Contains a Potential Tail-Dependent G-Quadruplex That Downregulates Gene Expression upon Oxidative Modification. ACS Chem Biol 13:2577-2584
Fleming, Aaron M; Ding, Yun; Burrows, Cynthia J (2017) Sequencing DNA for the Oxidatively Modified Base 8-Oxo-7,8-Dihydroguanine. Methods Enzymol 591:187-210
Fleming, Aaron M; Zhu, Judy; Ding, Yun et al. (2017) 8-Oxo-7,8-dihydroguanine in the Context of a Gene Promoter G-Quadruplex Is an On-Off Switch for Transcription. ACS Chem Biol 12:2417-2426
Fleming, Aaron M; Ding, Yun; Burrows, Cynthia J (2017) Oxidative DNA damage is epigenetic by regulating gene transcription via base excision repair. Proc Natl Acad Sci U S A 114:2604-2609
Ding, Yun; Fleming, Aaron M; Burrows, Cynthia J (2017) Sequencing the Mouse Genome for the Oxidatively Modified Base 8-Oxo-7,8-dihydroguanine by OG-Seq. J Am Chem Soc 139:2569-2572
Fleming, Aaron M; Burrows, Cynthia J (2017) 8-Oxo-7,8-dihydro-2'-deoxyguanosine and abasic site tandem lesions are oxidation prone yielding hydantoin products that strongly destabilize duplex DNA. Org Biomol Chem 15:8341-8353
Fleming, Aaron M; Burrows, Cynthia J (2017) 8-Oxo-7,8-dihydroguanine, friend and foe: Epigenetic-like regulator versus initiator of mutagenesis. DNA Repair (Amst) 56:75-83

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