We hypothesize that a group of oxidative DNA damages, including 8-oxoguanine, formamidopyrimidines and tandem lesions, play important roles in the etiology of human cancer via the mutagenic and genotoxic effects of these lesions in certain important sequences of cancer genes. In this application, we propose to study the biological effects of a subset of these lesions. We have three specific aims.
In aim 1, synthesis and characterization of oligonucleotides containing tandem base lesions in DNA will be carried out. Tandem lesions formed by ionizing radiation, transition metal/H2O2, or other radical- mediated processes include abasic site adjacent to 8-oxoguanine, S- and R-8,5'-cyclo-2'- deoxyguanosine, and intrastrand vicinal guanine-thymine cross-link. We shall determine the structural effects of these damages in DNA by thermal melting, circular dichroism, and NMR.
In aim 2, single stranded plasmid or viral vectors containing these lesions will be constructed. Mutagenicity and repair studies of the site-specific lesions will be carried out in Escherichia coli and in mammalian cells. For example, we shall determine how mutagenicity of 8-oxoguanine might be influenced when it is present as part of a tandem DNA damage. We shall investigate if Fapy.dG, the guanine-thymine vicinal cross- link, S- and R-8, 5'-cyclo-2'-deoxyguanosine are mutagenic, genotoxic, or both in mammalian cells.
In aim 3, we shall construct duplex plasmid vectors with these lesions, which will be used to examine the extent of lesion bypass by extracts of normal and repair- or replication-altered mammalian cells (e.g., isogenic mouse embryonic fibroblasts and cells that express modulated levels of DNA polymerases encoded by REV1, REV3, RAD18, etc. genes). We hope to contribute toward a deeper insight of the effects of oxidative DNA damages.
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