CHEMICAL CARCINOGENESIS AND CELL PROLIFERATION

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The objective of this project is to characterize the relationship between carcinogen damage to nuclear DNA and the process of DNA replication during the S phase of the cell cycle. We have observed that DNA associated with replication forks is preferentially methylated by chemical carcinogens, suggesting that DNA replication may affect the distribution of carcinogen bound to DNA. We now propose to determine whether this phenomenon can be observed and quantitated at the level of individual genes. With the help of antibodies that recognize specific DNA adducts, we will precipitate DNA fragments containing lesions and determine whether this DNA fraction is enriched in gene sequences that were replicating at the time of carcinogen treatment. Antibodies to carcinogen adducts in DNA, together with proteins that bind to single-stranded DNA regions, will be used with the electron microscope to determine the distribution of adducts and the presence of daughter strand gaps at DNA replication forks. We will compare such distributions with the biochemical pattern of inhibition of DNA synthesis after carcinogen treatment and the effects of carcinogens on the distribution of active replicons in synchronized cells, as visualized by fiber autoradiography. Such approach will provide new insights into mechanisms of DNA chain elongation, by-pass of DNA lesions and recovery from the overall inhibition of DNA replication. The biochemical characterization of the inhibition of DNA replication will rely on the study of distributions of nascent DNA molecules in alkaline sucrose gradients following carcinogen treatment of asynchronous, as well as synchronized cells. The latter will be particularly useful in studying the process of maturation and joining of intermediates of DNA replication in damaged S phase cells (i.e., post-replication repair). The results of these experiments will be analyzed in conjunction with data on the effects of the chemical carcinogens on cell cycle parameters, such as rate of entry of cells into S and M phases and prolongation of the replicative period.