Abstract 1035 Biological and molecular consequences of oxidative damage on non-B DNA-induced genetic instability

DNA can adopt several alternative structures (i.e., non-B DNA) including intramolecular triplexes (H-DNA). H-DNA forming-sequences co-localize with mutation hotspots in human cancer genomes and induce genetic instability in mammalian cells and in mice, predominantly through the generation of mutations such as deletions. Some non-B DNA structures (e.g. hairpins and G4-DNA) have been found to be targets for base oxidation under conditions of oxidative stress. Because of the structural alterations in H-DNA, we hypothesize that H-DNA-forming regions may be more susceptible to oxidative damage, leading to increased genetic instability in human cells. The objective of this research is to determine the extent to which DNA damage accumulates in H-DNA-forming sequences and the subsequent impact of DNA damage on H-DNA structure formation, stability, processing, and genomic instability. Using an immuno-slot blot assay, we show that oxidative damage resulted in more 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) adducts, a biomarker for oxidative DNA damage, in H-DNA-forming sequences compared to its control B-DNA counterpart of the same length and GC content. Second, we examined the processing and mutagenic potential of H-DNA-forming sequences in the presence of oxidative DNA damage. Damaged mutation-reporter DNA containing either an H-DNA-forming sequence or a control B-DNA-forming sequence was transfected into mammalian cells and screened for mutations. Our results indicate that increasing levels of oxidative damage attenuated the fold increase in H-DNA-induced mutation frequency over control B-DNA in mammalian cells. Furthermore, analyses of the mutation spectra suggest that the oxidatively-damaged H-DNA-containing template was processed differently by the cells compared to that of the H-DNA-containing template without oxidative damage. Finally, thermal denaturation experiments show that the presence of a single 8-oxodG destabilizes the H-DNA structure. Together, these results suggest a new model of genetic instability whereby H-DNA-forming regions are hotspots for DNA damage resulting in altered biological processing of H-DNA. This research was supported by a department grant from the Robert A. Welch Foundation [AF-0005] (MZF), the National Institutes of Health/NCI (KMV), the National Institutes of Health/NIEHS (KMV and MZF), and the Garey Endowed Chair in Chemistry at Southwestern University.