The broad objective of this proposal is to understand the molecular details of DNA repair, an important defense mechanism against damage to DNA, in the context of DNA packaging and gene transcription in chromatin. Such insults result from a wide variety of environmental agents, such as ultraviolet (UV) radiation and chemical carcinogens. UV radiation and dimethylsulfate will be used as prototype environmental agents for studies on nucleotide excision repair (NER) and base excision repair (BER), respectively. We will examine the relationship between DNA repair, transcription and chromatin structure of different classes of genes in well-defined nucleosome (or transcription factor) complexes in vitro, as well as in intact yeast and mammalian cells. Repair of an RNA pol II gene promoter will be examined in a mouse viral gene promoter (LTR), which is induced upon binding of a hormone-receptor. The major form of UV damage in DNA (csCTD) has been synthesized and will be incorporated into specific sites of the LTR for studies on the effect of csCTDs on receptor binding. This sequence will also be bracketed by nucleosome positioning elements and packaged into a nucleosome for studies on the effect of csCTD orientation in a nucleosome on NER efficiency in Xenopus extracts. G::U mismatches will also be synthesized into the LTR to examine the effect of protein binding on BER efficiency, using purified human and BER proteins and mammalian cell extracts. Finally, repair of CTDs and N-methyl purines (NMP) will be examined in yeast ribosomal RNA genes (rDNA). Unlike pol II genes, repair of UV photoproducts in active and inactive rDNA is inefficient in mammalian cells, but is rapid in yeast. We will induce different fractions of transcriptionally active ribosomal chromatin in yeast, by varying growth conditions, and examine repair of CTDs in each fraction. We will determine if efficient repair of active rDNA also occurs for NMPs, which are rapidly removed from other genomic sequences by the smaller BER proteins. Thus we will examine the effects of gene expression and changes in local chromatin structure on the efficiency of DNA repair. Since these lesions may alter the expression of specific genes required for establishing the neoplastic phenotype, these studies should provide insight into the cell's defense mechanism for resisting transformation by environmental carcinogens.
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