Cellullar Response to DNA Adducts

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DESCRIPTION (provided by applicant): DNA synthesis across unrepaired DNA damage, known as translesion DNA synthesis (TLS), is one of the major mechanisms responsible for chemical mutagenesis; in humans this process is associated with the development of cancer and age-related degenerative diseases. The goal of this research is to elucidate the mechanisms of TLS as they occur in human cells. Several TLS-specialized DNA polymerases (pol), such as pol h, pol k, pol i, pol z and REV1, have been discovered recently; we will determine the role of these enzymes in TLS in human cells. Two exocyclic propanodeoxyguanosine (PdG) DNA adducts and 1,N6-ethenodeoxyadenosine (edA) will be employed as target lesions. We have established the genotoxicity of these adducts in human cells, using a unique shuttle vector system developed in my laboratory. To probe the mechanism for TLS in cells, primer extension studies with purified polymerases will be first conducted to determine their efficiency, fidelity, and coding specificity in vitro. By comparing in vivo and in vitro results, candidate polymerase(s) responsible for TLS events in cells will be identified. The expression of the candidate polymerase gene(s) in human cells will be silenced by RNA interference technology. Such "engineered" cells are then used as hosts for genotoxic analyses. By comparing TLS events in "wild type" and engineered cells, the role and function of candidate polymerase(s) in TLS can be assessed. Extracts of engineered cells will be used to replicate modified plasmid in vitro for biochemical studies. The combination of in vitro TLS studies, in vivo assays for genotoxicity, and RNA interference technology will provide novel insights into the fundamental role and function(s) of TLS polymerases and their contribution to chemical mutagenesis in human cells.