The SV40 large T antigen (T Ag) has the ability to transactivate a variety of cellular and viral promoters. However, it is not known whether the ability of T Ag to activate transcription contributes to cell growth and transformation. In the previous grant period, we used the temperature sensitive cell line, ts13, to address this question. The ts13 cell line contains a point substitution mutation in TAF1, an essential component of the core transcription complex TFIID. Upon shift from the permissive temperature to the restrictive temperature, ts13 cells undergo a growth arrest in the G1 phase of the cell cycle. Furthermore, transcription from a limited number of promoters is significantly reduced in ts13 cells at the restrictive temperature. T Ag is capable of overriding the growth arrest as well as the transcriptional defects in the ts13 cell line at the restrictive temperature. Our efforts led to the unexpected findings that the growth arrest and perhaps the transcription defects in ts13 cells reflected activation of a DNA damage checkpoint and that T Ag can inactivate or bypass both of these DNA damage signals. We observed that T Ag's p53 and pRb binding domains override the growth arrest and the DNA binding domain (DBD) rescues the transcriptional defects. These two effects of T Ag on ts13 cells may reflect two independent mechanisms that T Ag has evolved to overcome DNA damage checkpoints. We propose that the TAF1 defect triggers a DNA damage response that mediates growth arrest through activation of p53. In addition, the transcription defects may also reflect a DNA damage response. To address these issues, we propose the following Specific Aims: 1. Determine if the ability of the T Ag DBD to rescue the transcriptional defect in ts13 cells is due to interference with a DNA damage checkpoint or rescue of transcriptional activation. 2. We will explore the specific contribution of TAF1 to the DNA damage signal by examining the role of its histone acetyl transferase (HAT) and ubiquitin-activating/conjugating (Ubac) activities. 3. Determine the DNA damage factors activated in ts13 at the restrictive temperature and contrast with activation of the intra-S phase and double strand break DNA damage checkpoints. We will examine the ability of T Ag to perturb recruitment of factors to sites of DNA damage. 4. Generate a mouse model of the ts13/TAF1 defect to explore the genetic interaction between transcription and DNA damage checkpoint response.
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