From studies with ultraviolet (UV) or ionizing radiation, it is clear that DNA is a 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 killings since generally some type of processing (e.g. repair or bypass) is required in order for such lesions to be """"""""fixed"""""""" and thus result in mutagenesis, carcinogenesis or lethality. This project will examine this processing by examining DNA replication after insult of mammalian cells with ultraviolet light (UV). DNA replication was chosen since existing data suggest that it may play a crucial role in the processing of DNA lesions. UV was selected as the carcinogen/mutagen because the yield stability and repairability of UV induced DNA lesions is fairly well understood, and because it is possible to selectively remove (by enzymatic photoreactivation) pyrimidine dimers (the most frequent and probably most important UV induced lesions). The time course of the effects of UV on DNA synthesis will be examined in intact cells and in permeabilized cells (cells rendered permeable to various metabolites) by using DNA fiber autoradiography as well as conventional kinetic incorporation studies. This study will include an examination of the molecular events responsible for both initial depression in the rate of DNA synthesis and the eventual recovery in the rate. Emphasis will be placed on elucidating the molecular processes involved in the recovery phase since these are the processes suspected of being carcinogenic and/or mutageic. It is our hope that this project will answer some very basic questions concerning the manner in which the replicative complex handles and bypasses DNA lesions. At a minimum that data obtained should indicate whether it is worth continuing to examine the role of DNA replication in mutagenesis/carcinogenesis. Hopefully, the data will also result in information that can be used to better understand carcinogenesis and mutagenesis.
