The biological endpoints of DNA damage are initiated primarily by its effects on the DNA and RNA synthesis machinery. RNA polymerase arrest at or near a DNA damage site is thought to be a key event in transcription-coupled repair. We have recently shown that in vitro, certain types of ionizing radiation-induced DNA damages can be efficiently bypassed by RNA polymerases and produce mutagenic insertions or deletions in the resulting transcript (transcription mutagenesis). We believe that such events have potentially profound implications for both DNA repair pathways as well as the cellular mechanisms that lead to the production of mutant proteins. Whether or not the failure of a given DNA damage to block transcription elongation has important biological consequences is presently unknown. One of the major goals of this proposal is to test the hypothesis that transcription mutagenesis can occur in living cells. For these studies we will utilize both in vitro and in vivo transcription systems. We will determine the effects of the ionizing radiation-induced base damage products uracil, dihydrouracil, and 8-oxoguanine and the alkylating agent-induced base damage product 06methylguanine. An important criterion for selecting these DNA base damage products is that they also cause mutagenic nucleotide insertions when encountered by DNA polymerases. Specifically we will: (1) place these defined base damage products into DNA templates and determine the effects of such lesions on the prokaryotic transcription elongation machinery in vitro; (2) determine the ability of these base damage products to generate mutant transcripts and proteins in vivo in stationary cultures of E. coli with different DNA repair backgrounds; (3) place these base damage products into DNA templates and determine the effects of such damages on the mammalian transcription elongation machinery in vitro; and (4) determine the ability of these base damage products to generate mutant transcripts and proteins in vivo in non- replicating mammalian cells. The in vitro studies will provide a direct indication of whether or not RNA polymerase bypass of specific types of ionizing radiation-induced DNA damages occurs and, if bypass occurs, whether or not such an event results in mutagenic base insertions in the resulting transcript. The in vivo studies will address whether or not such mutagenic insertions occurring at the level of transcription can alter protein expression in a non-dividing cell.
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