The proposed studies address the basis for an unusual DNA sequence selectivity for damage produced by nitrosoperoxycarbonate (ONOOCO2-), a chemical mediator of inflammation. In a manner opposite to """"""""classical"""""""" one electron DNA oxidants, the reactivity of ONOOCO2- toward guanine (G) in DNA was observed to increase as a function of the calculated sequence-dependent G ionization potential. In contrast, G reactivity toward photooxidation decreases with increasing ionization potential. We propose to investigate this phenomenon to test several hypotheses: (1) that the behavior of ONOOCO2- is common to negatively-charged oxidants; (2) that the behavior reflects preferential oxidation of G's that are more solvent exposed; and (3) that the initial one-electron oxidation event determines the selectivity of G oxidation by ONOOCO2- and other oxidants. The results will provide new insights into the mechanistic link between inflammation and cancer by revealing the chemical basis for the location of DNA damage and subsequent mutations.
Aim 1 : Define the DNA sequence selectivity of carbonate radical anion (CO3.), ONOO-, ONOOCO2- and related oxidants. We will extend preliminary studies to include a range of DNA sequences (oligos, 32P-DNA fragments and genomic DNA) and ONOOCO2- concentrations. We will also test the hypothesis that the reactivity of a G in DNA is influenced by the charge of the oxidant by comparing the sequence selectivity of ONOOCO2- to that for a variety of negatively-charged, neutral and positively-charged oxidants.
Aim 2 : Define the basis for the sequence selectivity of CO3., ONOO- and ONOOCO2-. First, we test the hypothesis that the sequence selectivity of G oxidation arises at the first step of the oxidation reaction and not as a result of later product formation. We then proceed to test the hypothesis that solvent exposure governs the reactivity of G's toward ONOOCO2- and other oxidants by: (1) comparing sequence selectivity in single- and double-stranded oligos; and (2) assessing the imino proton exchange rates of G's in different DNA sequence contexts.
Aim 3 : Compare the sequence selectivity of DNA damage chemistry for CO3. and ONOOCO2-. To complement Aims #1 and #2, we will test the hypothesis that the products of G oxidation vary as a function of sequence context. First, we define the proportions of base and deoxyribose oxidation in plasmid DNA treated with the oxidants and then proceed to investigate the role of DNA sequence context in the spectrum of G oxidation products. Guanine lesions in the oligos will be characterized by LC-MS for single G sequences and by exonuclease digestion/MALDI-MS for localizing damage in sequences with multiple G's.
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