Mechanisms of Nucleic Acid Oxidation and Cross-linking

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With the support of the Organic Dynamics Program in the Chemistry Division, Professor Cynthia J. Burrows of the Chemistry Department at the University of Utah will formulate a detailed molecular picture of how DNA is oxidized in the presence of reactive species such as protein, polyamine and catecholamine nucleophiles. A complete mechanistic picture of how such adducts form is important in order to understand the fundamental processes in damage to genomic DNA. Studies outlined in this proposal build upon recent results showing that 8-oxo-7, 8-dihydroguanosine is sensitive to further oxidation by one-electron oxidants leading to a quinonoid intermediate that is nucleophilically trapped to generate hydantoin products. Recent progress has shown that other oxidized bases are subject to the same facile oxidation/nucleophilic trapping mechanism. Experiments proposed include NMR and LC/MS characterization of amino acid and polyamine adducts to nucleosides and to DNA oligomers, and investigation of their mechanisms of formation and chemical stability. Methods will be developed to understand the chemical structures responsible for DNA-protein cross-linking via both DNA oxidation and protein oxidation. Adducts of phenols and catechols to guanosine in DNA will be prepared and analyzed for their ability to mediate further oxidative damage to DNA. A major part of the new research will focus on characterization of reactive quinonoid intermediates in 8-oxoguanosine and uric acid oxidation. Nucleosides and synthetic oligonucleotides will be studied by stopped-flow spectrophotometry and spectroelectrochemistry to decipher individual steps in the formation and decay of the quinones. Mechanistic parallels are proposed between purine oxidation and the pterin and flavin redox cofactors, leading to the transformative hypothesis that RNA damage products were ancestral to the evolution of nucleotide coenzymes.This research by Professor Burrows of the University of Utah will include the training of undergraduate and graduate students who will be well versed in mechanistic organic chemistry, gain biotechnical skills in the manipulation of nucleic acids and proteins, and contribute to our molecular understanding of DNA damage, which underlies processes leading to aging, cancer, and neurological disorders. A major new effort in this project is the development of a library of DNA adducts that will be prepared by undergraduate research students. The organic synthesis is straightforward and appropriate for students with little or no prior research experience. Students will prepare new nucleoside adducts by selecting from a wide variety of commercially available nucleophiles (e.g. primary amines such as amino acids), then purifing 5 mg of each adduct by HPLC, and loading them onto 96-well plates. Students will then analyze the plates by UPLC/ESI-MS and submit them for a battery of screening tests for biological activity at the University of Utah?s new core facility for biomolecular screening. Undergraduate researchers are recruited from the University?s ACCESS program, which fast-tracks high achieving women high school students into research labs in their freshman year. Professor Burrows is the Director of the Chemistry-Biology Interface Training Program from which minority undergraduate researchers will be recruited.