Reactive oxygen species (ROS) in the cell lead to DNA damage including base lesions that are mutagenic if unrepaired. The molecular events leading to mutagenesis now provide a direct link between oxidative DNA damage and cancer. An understanding of the molecular basis of carcinogenesis is the foundation of prevention and treatment. The unusual heterocycles 5-guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp), whose structures were recently assigned in this laboratory, are major products of guanosine and 8-oxo-7,8-dihydroguanosine (OG) oxidation by a wide variety of (ROS) including singlet oxygen and peroxyl radicals. In the past funding period, these laboratories have investigated the chemical mechanism of formation of Gh and Sp, developed methods of generating highly pure synthetic oligodeoxynucleotides containing these lesions, studied misinsertion of nucleotides opposite the lesions as well as repair by base excision repair (BER) enzymes, and observed a 99 percent mutation rate of both lesions in an in vivo assay. The recent detection of Sp in repair-deficient bacterial cells increases the relevancy of these lesions to oxidative damage and disease.
The aims of the next period seek to understand the molecular basis for the extremely high mutation rates and the unusual G to C mutations observed with Gh and Sp. Other oxidized purines including OG will be studied for comparison when appropriate. Specifically, this work will (1) determine the structures of Gh and Sp in duplex DNA using NMR and x-ray crystallographic techniques, (2) examine misinsertion of nucleotides opposite the lesions with Y-family (lesion bypass) DNA polymerases and investigate sequence-dependent frameshift mutagenesis with eukaryotic polymerases, (3) study the molecular mechanisms of repair of Gh and Sp lesions using BER glycosylases, and (4) study in vivo mutagenesis using site-specifically incorporated lesions in DNA plasmids. All of the specific aims require continual refinement of our synthetic procedures for generation of nucleoside standards, nucleotide triphosphates and oligonucleotide substrates as well as mass spectrometric characterization of the lesions. Collaborators will assist with NMR and x-ray crystallography, and in vivo mutagenesis studies in E. coli. This laboratory will continue to aid collaborators who are developing analytical methods to determine the cellular levels of the Sp and Gh lesions. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
2R01CA090689-05A1
Application #
7144704
Study Section
Cancer Etiology Study Section (CE)
Program Officer
Okano, Paul
Project Start
2001-04-01
Project End
2010-05-31
Budget Start
2006-07-01
Budget End
2007-05-31
Support Year
5
Fiscal Year
2006
Total Cost
$304,398
Indirect Cost
Name
University of Utah
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
009095365
City
Salt Lake City
State
UT
Country
United States
Zip Code
84112
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
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
Fleming, Aaron M; Burrows, Cynthia J (2017) Formation and processing of DNA damage substrates for the hNEIL enzymes. Free Radic Biol Med 107:35-52
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

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