Cellular responses to oxidative stress are a critical line of defense against reactive oxygen species that alter the structure of DNA bases leading to mutagenesis. Errors in the DNA sequence and impaired repair or signaling of DNA damage underlies numerous disorders including cancer, Alzheimer's disease and metabolic disorder. This program has uncovered the chemistry and biochemistry of hyperoxidized guanines including spiroiminodihydantoin, guanidinohydantoin and 2-iminohydantoin. These heterocyclic lesions form in G-rich sequences particularly those capable of folding into a G-quadruplex (G4). The processing of hydantoin lesions in double-stranded DNA vs. G4-DNA appears to be quite different with various members of the NEIL family participating. We hypothesize that the hydantoins in G-quadruplex structures such as promoter sequences function as a sensing mechanism for up-regulation of repair enzymes involved in oxidative stress. This project aims to examine the structures and stabilities of G-quadruplex-forming DNA sequences as a function of oxidative stress, and to examine their binding interactions and glycosylase kinetics with base excision repair proteins. The approach involves synthesis of oligodeoxynucleotides containing site-specifically incorporated G oxidation (8-oxoguanosine or the hydantoins) and to examine the thermal stability, circular dichroism spectra, and the ability to bind to NEIL1-3 or NTHL1. In addition, the ability of damage- containing G4-DNA to induce or inhibit transcription will be studied in a reporter plasmid assay. Innovative concepts and methods include the study of numerous predicted G4 sequences that may play regulatory roles in BER enzyme expression and the suggestion that oxidized bases in DNA could serve 'epigenetic-like' roles in transcription. Novel methods of studying G- quadruplex structure include the use of an ion channel protein, alpha-hemolysin, as a single- molecule reporter of structure for assaying a collection of equilibrating species. The significance of this work lies in uncovering the molecular basis for diseases related to oxidative stress, with a particular focus on the chemistry of the oxidatively damaged DNA and how it triggers structural reorganization in DNA to choreograph downstream events leading to mutation or repair.

Public Health Relevance

Free radicals and oxidative stress are contributors to cancer and other age-related disorders. This work will examine the chemistry of oxidation of the DNA base guanine in guanine-rich sequences such as those found at the ends of chromosomes and in gene promoter regions. It will test the hypothesis that changes in DNA folding induced by oxidative stress play a role in regulating protein expression.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA090689-15
Application #
8985659
Study Section
Synthetic and Biological Chemistry A Study Section (SBCA)
Program Officer
Okano, Paul
Project Start
2001-04-01
Project End
2020-04-30
Budget Start
2016-05-01
Budget End
2017-04-30
Support Year
15
Fiscal Year
2016
Total Cost
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
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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