Clinical and experimental data suggest that chronic infection and inflammation are associated with an increased risk for developing human cancers. In response to inflammation and infection, the cellular production of diverse reactive oxygen (ROS) and nitrogen (RNS) species is enhanced. There is recent evidence that carbonate radical anions may play an important role in damaging biological macromolecules, especially proteins and lipids, in cellular environments subjected to oxidative stress. However, little is known about the interactions of carbonate radicals with DNA. The central hypothesis of this project is that carbonate radical anions site-selectively oxidize guanines by a one-electron transfer process, thus resulting in the formation of guanine radicals in DNA. We have shown that the guanine radicals in double stranded DNA are sufficiently long-lived (about seconds) to react with various other radical species such as nitrogen dioxide radicals to form site-specific nitroguanine adducts, as well as decomposition products of the latter.
Specific aim 1 is to establish the pathways of reaction of these guanine radicals in oligonucleotides of defined base sequence and composition. The concentrations of the guanine radicals in double stranded DNA will be monitored in real time employing laser excitation transient absorption spectroscopy techniques. The further oxidation of these guanine radicals by carbonate radicals and their reactions with superoxide radical ions will be monitored as a function of time, together with the kinetics of appearance of chemical reaction products after reaction time intervals defined by kinetic flow-quench methods. The chemical nature of adducts resulting from the reactions of carbonate radicals will be identified using standard chemical and analytical techniques.
In specific aim 2, the reactivities of guanines in runs of two, three, and four guanines in structurally similar oligonucleotides will be assessed to determine if their reactivities are enhanced by other flanking Gs as predicted on theoretical grounds, and to determine if runs of guanines can constitute hotspots of oxidative DNA damage initiated by carbonate radicals.
In specific aim 3 the reactivities of guanine radicals in DNA with nitrogen dioxide will be assessed along the lines described for the carbonate radicals in specific aims 1 and 2, and the reaction products will be identified. A photochemical method will be employed to synthesize site-specifically modified oligonucleotides with well-defined single nitroguanine/xanthine lesions.
In specific aim #4, the susceptibilities of these nitroguanine/xanthine lesions to excision by selected base excision repair enzymes, and their mutagenic potentials in site-directed mutagenesis experiments in mammalian cells will be assessed.
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