The breast cancer tumor suppressor gene, BRCA1, has been shown to play a critical role in response to DNA damage. Previously, RNA Polymerase II (RNAPII) was shown to be both phosphorylated and ubiquitinated in response to DNA damage. Data from our lab has shown that BRCA1 can ubiquitinate RNAPII in cells, and this is stimulated by DNA damage. However, other ubiquitination targets of BRCA1 in response to DNA damage are currently unknown. We hypothesize that a RNAPII associated mechanism is responsible for an inhibition in the repair of DNA lesions and that the BRCA1 ubiquitination activity plays a key role in the suppression of this mechanism. We also predict that BRCA1 ubiquitinates a number of other factors in response to DNA damage.
Specific aims : (1) Determine the role of BRCA1 and the inhibition of transcription in DNA damage repair using a novel transcriptional assay and a newly developed cell line capable of inducing DNA damage at a defined locus. (2) Establish ubiquitination targets of BRCA1 in response to DNA damage in vivo, determine if BRCA1 directly ubiquitinates the identified targets in vitro, and assess the identified targets in vivo role in response to DNA damage. Study design: 1a: A novel transcriptional assay will be used to assess the roles of BRCA1 and the inhibition of transcription in DNA damage repair. 1b: A new cell line that is capable of inducing a double strand DNA break (dsDNA) at a defined locus will be used along with ChIP assays to determine what repair factors are present at the dsDNA break in the presence and absence of a) BRCA1 and b) an active transcription process. 2: A cell line that expresses a double-tagged ubiquitin will be used to purify ubiquitinated proteins in response DNA damage and proteins found to be specifically ubiquitinated by BRCA1 will be identified by mass spectrometry. Their role in the response to DNA damage will be investigated by knockdown and/or overexpression analysis. The long-term implications of this project are to further elucidate the mechanisms by which BRCA1 acts in the DNA damage response by revealing its role. This project will also discover the biochemical mechanisms of BRCA1 action. In addition, other players, independent of transcription, that are targets of BRCA1 in response to DNA damage will be revealed. The improper repair of DNA damage is a major contributor to the formation of cancer cells. A better understanding of this basic mechanism has the potential to lead to new avenues of drug design and other cancer therapies. ? ? ?
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| Arora, Mansi; Zhang, Jie; Heine, George F et al. (2012) Promoters active in interphase are bookmarked during mitosis by ubiquitination. Nucleic Acids Res 40:10187-202 |
| Heine, George F; Horwitz, Andrew A; Parvin, Jeffrey D (2008) Multiple mechanisms contribute to inhibit transcription in response to DNA damage. J Biol Chem 283:9555-61 |
