Mechanism of Eukaryotic Environmental Mutagenesis

Research funded by this grant has yielded important new insights into the mechanism and biological roles of the specialized DNA polymerases that carry out translesion synthesis (TLS). Certain TLS polymerases are able to copy quite accurately over particular "cognate" lesions but others, notably Rev1 and Pol (Rev3/Rev7) in eukaryotes, participate in a mutagenic branch of TLS that is responsible for most of the mutations that result from exposure to radiation and DNA damaging chemical agents. In addition to revealing the critical importance of certain protein-protein interactions in TLS, our studies suggest that TLS polymerases may also play important roles in the mitochondria and provide striking evidence suggesting that drugs that could suppress the action of Rev1/Pol-dependent TLS could have very beneficial applications for human health. The proposed experiments will yield new insights into the molecular mechanism of TLS and are designed to identify a new class of drug that acts by inhibiting TLS. They will also evaluate the roles of TLS polymerases in mitochondria and further characterize the roles of TLS polymerases in tumors that are undergoing chemotherapy in vivo. Experiments are proposed involving parallel approaches in both yeast and mammalian cells that should allow us to gain fundamental knowledge into the molecular details of Rev1/Pol-dependent TLS, while at the same time allowing us to develop high-throughput fluorescence polarization assays for screening for small molecules that disrupt Rev1-Rev7-Rev3 interactions. We also plan to test whether Rev1/3/7 function can be disrupted by ¿-helical stapled peptides and by cutting-edge siRNA-based approaches. There are presently only three papers in the literature describing the roles of TLS polymerases in the mitochondria, however our results during the past progress period strongly support the possibility that TLS polymerases play additional key role in DNA damage tolerance in the mitochondria, an important topic because of the many human diseases associated with mutations in mitochondrial DNA. We will determine which TLS polymerases are important for mitochondrial DNA mutagenesis in response to selected DNA damaging agents.

Results during the past progress period have yielded new insights into the importance of TLS in tumors undergoing chemotherapy, and we will continue to study TLS in this important biological context and we will continue to use mouse models to investigate the in vivo roles of Rev1 and Pol in mitochondrial and nuclear mutagenesis. We will determine the nature of the Rev1/3/7-dependent mitochondrial and nuclear mutations occurring in mouse models of cancer during DNA damaging chemotherapy and investigate how key mutations associated with cancer progression and DNA repair/checkpoints affect a cell's ability to use Rev1/3/7 dependent TLS to withstand DNA damage and to mutate in response to such damage.