Cisplatin is a probable human carcinogen. Platinum compounds such as oxaliplatin have shown promise for the treatment of cisplatin resistant tumors, but their potential mutagenicity is unknown. The mutagenicity of these compounds is likely to be determined in part by the DNA polymerase(s) which catalyze error-prone translesion synthesis past Pt-DNA adducts. The identity if these polymersases is not currently known, but pol beta, pol zetu, and pol eta have all been shown to perform translesion synthesis past Pt-DNA adducts in vitro. Cell lines exist which under- or over-express each of these polymersases. Thus, we plan to determine the effects of pol beta, pol zetu, and pol eta expression on the mutagenicity and cytotoxicity of cisplatin and oxaliplatin adducts (Aim 1). An analysis of the conformational differences of the adducts formed by these compounds is likely to be essential for understanding the mechanisms which determine their mutagenicity and efficacy. Thus, we plan to use a combination of NMR and molecular modeling to characterize the adducts formed by these compounds (Aim 2). Detailed information on the efficiency and fidelity of translesion synthesis by purified DNA polymerases is important for confirming the contribution of those polymersases to error-prone translesion synthesis in vivo (Aim 1) and for modeling their interaction with the adducts (Aim 4). Thus, the efficiency and fidelity of translesion synthesis past cisplatin and oxaliplatin adducts by pol beta, pol zetu, and/or pol eta will be determined in vitro (Aim 3). It is likely that the conformation and mutagenicity of Pt-DNA are affected by their binding to the active site of the polymerases involved in error-prone translesion synthesis. Based on the conformational data on the adducts (Aim 2) and the known crystal structures of pol beta, molecular dynamics simulations will be used to model the interactions of pol beta with each of the adducts (Aim 4). This project will define the role of pol beta, pol zetu, and pol eta in error-prone replicative bypass of Pt-DNA adducts. It will also result in models of Pt-DNA adduct conformation and polymerase-adduct interaction that will be useful in understanding the mechanism(s) of mutagenesis by these and other Pt complexes.
Showing the most recent 10 out of 18 publications
