Diepoxides alkylate the N7 position of deoxyguanosine residues on opposite strands of DNA, leading to interstrand cross-linking. Such bifunctional lesions can have either antitumor potential or merely cause cancer. The mechanisms by which different cross-linkers elicit profoundly different biological responses remain unclear. The proposed research will examine the mediation of diepoxidecross-linking reactions by cellular proteins and will also investigate the structure-function relationship of diepoxide cross-linking reactions with the long-term goal of elucidating the mechanism of carcinogenicity versus anti-cancer chemotherapeutic potential. ? ? DNA is complexed with histone proteins in the cell nucleus to form chromatin, which modulates the reactivity of some external agents with DNA. However, we have shown previously that DNA within the nucleosomal core particle remains remarkably accessible to cross-linkers. We will continue our studies on whole cells using a Ligation-Mediated Polymerase Chain Reaction to define further the sites of attack and relative efficiencies for diepoxyalkanes within cellular DNA in order to provide information about the genomic sites of cross-linking for agents with differing biological activities. ? ? Overexpression of the DNA-binding protein HMG1 in breast cancer cells leads to an enhancement of the therapeutic efficacy of cisplatin. HMG1 may also recognize bending induced by diepoxide cross-links. Diepoxide-cross-linked DNA will be monitored for bending through native polyacrylamide gel electrophoresis (PAGE) and binding to HMG1 by gel shift assays. These studies will allow the exploration of the relationship between the geometry of a DNA cross-linked lesion and recognition by HMG proteins. This information has the potential to impact drug design: future agents could be designed to trigger HMG binding to specific DNA sequences and thus induce cancer cell death. ? ? Denaturing PAGE will also be used to characterize DNA reaction products of epichiorohydrin to elucidate the impact of a three-carbon chain length in cross-linking.
