DNA REPAIR IN A HORMONE-RESPONSIVE GENE

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The broad objective of this proposal is to understand the molecular details of an important defense mechanism against phenotypic changes induced by insults to DNA in mammalian cells. Such insults result from a wide variety of environmental agents, such as ultraviolet (UV) radiation and chemical carcinogens. Since repair of the majority of these insults occurs via the same mechanism, UV radiation will be used as the prototype environmental agent for these studies. This proposal is designed to examine the relationship between DNA repair efficiency, transcriptional activity and chromatin structure of two different classes of mammalian cell genes. The relationship to RNA polymerase II expression will be examined in an isogenic mouse L cell line containing the herpes simplex virus thymidine kinase (tk) gene coupled to the mouse mammary tumor virus long terminal repeat (LTR). One and a half copies of this "LTL" construction is stably integrated into the genome of these cells and the expression of the tk gene is totally dependent on the presence of glucocorticoid hormone. We will use both Southern blot and Northern blot techniques to follow repair of UV damage in the LTL locus having the following properties: (1) inactive and packaged into a bulk chromatin structure; (2) inactive and packaged into a "transcriptionally poised" chromatin structure; and (3) actively transcribing. RNA polymerase I genes, having these same features, will be studied in mouse Friend cells. The ribosomal RNA genes of these cells will be fractionated into transcriptionally active and inactive forms using restriction enzyme digestion of nuclei and separation of the two forms of rDNA on preparative gels. Finally, we will use a simple yeast plasmid, containing an inducible gene and a constitutively expressed gene, as a model chromatin substrate in yeast cells to study specific feature of UV damage and repair of active genes in more detail. These studies will involve indirect end-labeling coupled with specific cleavage of the plasmid DNA at UV photoproducts by T4 endo V. Thus, we will examine the effects of both gene expression and changes in local chromatin structure on the efficiency of removal of DNA lesions. Since these lesions may alter the expression of specific genes required for establishing the neoplastic phenotype, these studies should provide valuable insight into the cell's defense mechanism for resisting neoplastic transformation by environmental carcinogens.