The goal of this proposal is to investigate transcriptional mutagenesis (TM) in a mammalian cell culture system, with application to a biologically relevant endpoint: the expression of a mutant Ras protein as a critical event in cancer development. Some of the most frequently occurring spontaneous types of damage (cytosine deamination to uracil, for example) are efficiently bypassed by RNA polymerase in vitro, potentially directing the incorporation of an incorrect nucleotide opposite the DNA lesion, leading to mutant transcripts and ultimately mutant proteins with biological effects. While an understanding of TM is emerging from studies in prokaryotic systems, much less is known about its occurrence in mammalian cells.
The specific aims proposed here are to (1) generate Ras expression constructs containing site-specific DNA damage that, if bypassed by RNA polymerase, will yield a transcript encoding a mutant Ras protein, (2) develop a mammalian cellular system in which to assess the effects of wild type or uracil-containing Ras TM constructs an subsequent signaling events related to tumorigenesis, and (3) characterize the effects of transcriptional processing of uracil-containing Ras constructs in cells with normal or compromised DNA repair capabilities.
| Saxowsky, Tina T; Meadows, Kellen L; Klungland, Arne et al. (2008) 8-Oxoguanine-mediated transcriptional mutagenesis causes Ras activation in mammalian cells. Proc Natl Acad Sci U S A 105:18877-82 |
| Saxowsky, Tina T; Doetsch, Paul W (2006) RNA polymerase encounters with DNA damage: transcription-coupled repair or transcriptional mutagenesis? Chem Rev 106:474-88 |
