The project's long-term objectives are to help elucidate the molecular processes involved in carcinogenesis and mutagenesis.
Specific aims relate to how the DNA lesions are handled in various eukaryotic cells. From studies with ultraviolet (UV) radiation, ionizing radiation, and other mutagens/carcinogens it is clear that DNA is the critical target for physical and chemical carcinogens and mutagens. The mere existence of lesions in DNA may not, however, be sufficient to produce mutagenesis, carcinogenesis or cell killing, since generally some type of processing is required for such lesions to be """"""""fixed"""""""" and thus result in mutagenesis, carcinogenesis or cell killing. This project examines this processing by examining DNA replication after insult of eukaryotic cells with UV (254 or 320 nm) light as well as some other mutagens/carcinogens. DNA replication was chosen since existing data indicate that it plays a crucial role in the processing of DNA lesions. There are three general areas to be explored. First, our previous work has shown that exposure of mammalian cells to UV light results in the activation of alternative sites of replicon initiation. Such an activation would allow cells to replicate DNA that would normally be prevented from being replicated by the presence of blocking lesions. This would increase cell survival, but might also increase the incidence of mutagenesis or carcinogenesis since this process does not remove lesions, but does stimulate cells to replicate regions of DNA with non-instructive bases. By using DNA fiber autoradiography we plan to continue our work in this area and to expand our study to other cells lines. Second, using DNA fiber autoradiography, we plan to continue our examination of the effects of UV light and the rate and extent of DNA fork progression. From such studies we hope to be able to identify the lesion(s) most responsible for blocking DNA fork progression. Finally, exposure to UV appears to inhibit the activation of some replicon clusters. This process appears to occur for a longer time (after exposure to UV) than does blockage to DNA fork progression. Using alkaline step elution, we plan to examine this process in detail. Examination of these three areas should increase our understanding of how cells handle potentially mutagenic/carcinogenic lesions. This, in turn, should increase our understanding of the molecular processes involved in mutagenesis and carcinogenesis.
