The long-term goals of this project are to understand the specific mechanisms involved in the damaging interactions of chemical carCinogens with chromatin. An ultimate carcinogen, benzo(a)pyrene diol epoxide (BPDE), that is derived metabolically from the environmental pollutant benzo(a)pyrene, is used as a model Carcinogen of broad relevance. Previous work has established the importance of DNA damage as a pro-mutagenic insult, and has shown that both the production and repair of this damage are modulated by DNA sequence and chromatin structure. Modest alterations in binding have been described previously with reconstituted models of the basic level of chromatin structure, the nucleosome. However, recent demonstrations of large differences in the binding of BPDE to different genes in intact cells suggest that higher levels of chromatin structure must produce increased modulation of binding. A nucleosome reconstituted in vitro on the promoter region of the hamster APRT gene will be incorporated into chromatin models of increasing complexity by adding the linker histone (H1), using longer DNA substrates, and adding a key non-histone protein, HMG 14, that is implicated in gene expression. Importantly, the ionic conditions will be altered to promote formation of the 30 nm fiber, a compact form of chromatin seen both in vitro and in vivo. The various chromatin preparations will be modified with BPDE, and the distribution of BPDE-DNA adducts in the APRT sequences will be determined by a laser-induced strand scission method. Biochemical studies of nuclei and analyzed under conditions of 30nm fiber formation will also be carried out. The second major theme of the project is to understand the mechanisms by which certain transcription factors, particularly Sp1, bind to the extent to which this phenomenon may disrupt normal gene expression in mammalian cells. Sequence contexts in the APRT gene in which a BPDE adduct constitutes an illegitimate binding site for SP1 will be identified and the structural parameters that are important for this interaction will be analyzed. Expression systems will be devised in DNA repair-deficient Chinese hamster ovary cells in which two potentially important epigenetic effect of illegitimate binding of SP1 to BPDE-DNA adducts, molecular hijacking and illegitimate activation, will be assayed at the level of transcription expression.
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