Copper (Cu) and iron (Fe) are biologically important metals that may play a role in the endogenous oxidative DNA damage involved in aging and cancer. However, we know little about their true genomic targets or the full spectrum of DNA lesions they produce. We will address these problems with a systematic and quantitative analysis of Cu- and Fe-induced DNA damage at four levels: deoxyribose oxidation chemistry, DNA conformation, chromatin structure and nuclear architecture.
AIM 1 : Chemistry of Cu- and Fe-induced deoxyribose oxidation in DNA. The goal of these studies is to complement our understanding of metal-induced base oxidation by defining the chemistry of deoxyribose damage caused by Cu and Fe under conditions of varying metal, H2O2 and reductant concentration.
AIM 2 : Quantitation and sequence-selectivity of metal-induced DNA damage. We will determine the sequence-selectivity and proportions of metal-induced base and sugar lesions in DNA in vitro and in vivo as a function of metal, H2O2 and reductant concentrations.
AIM 3 : DNA supercoiling and metal-induced DNA damage. We will extend our preliminary studies in which we show that damage produced by Cu, but not Fe, is sensitive to superhelical tension, such as occurs during transcription. To compare in vitro and in vivo results, we will perform these studies in a common DNA sequence, the rRNA genes.
AIM 4 : Nucleosome structure and metal-induced DNA damage. We will define the effects of nucleosome structure on metal-induced DNA damage and explore the mechanistic basis for our preliminary observation that CuII/H2O2 produces more damage in nucleosomes than in naked DNA.
2 AIM 5 : Nuclear architecture and metal-induced DNA damage. We will define the location of Cu and Fe in cell nuclei by x-ray fluorescence microscopy and test the hypothesis that metal-induced DNA damage chemistry is unevenly distributed in the genome of whole cells.
AIM 6 : Preparation of monoclonal antibodies against deoxyribose oxidation products. We will raise monoclonal antibodies against a major product of deoxyribose oxidation: 3'-phosphoglycolate residues. As a complement to base damage antibodies, the phosphoglycolate antibodies will be used to characterize DNA damage products in Aim number 1 and to quantify deoxyribose degradation in Aims number 2-5.
