Yeast screens identify the RNA polymerase II CTD and SPT5 as relevant targets of BRCA1 interaction.
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BRCA1 has been implicated in numerous DNA repair pathways that maintain genome integrity, however the function responsible for its tumor suppressor activity in breast cancer remains obscure. To identify the most highly conserved of the many BRCA1 functions, we screened the evolutionarily distant eukaryote Saccharomyces cerevisiae for mutants that suppressed the G1 checkpoint arrest and lethality induced following heterologous BRCA1 expression. A genome-wide screen in the diploid deletion collection combined with a screen of ionizing radiation sensitive gene deletions identified mutants that permit growth in the presence of BRCA1. These genes delineate a metabolic mRNA pathway that temporally links transcription elongation (SPT4, SPT5, CTK1, DEF1) to nucleopore-mediated mRNA export (ASM4, MLP1, MLP2, NUP2, NUP53, NUP120, NUP133, NUP170, NUP188, POM34) and cytoplasmic mRNA decay at P-bodies (CCR4, DHH1). Strikingly, BRCA1 interacted with the phosphorylated RNA polymerase II (RNAPII) carboxy terminal domain (P-CTD), phosphorylated in the pattern specified by the CTDK-I kinase, to induce DEF1-dependent cleavage and accumulation of a RNAPII fragment containing the P-CTD. Significantly, breast cancer associated BRCT domain defects in BRCA1 that suppressed P-CTD cleavage and lethality in yeast also suppressed the physical interaction of BRCA1 with human SPT5 in breast epithelial cells, thus confirming SPT5 as a relevant target of BRCA1 interaction. Furthermore, enhanced P-CTD cleavage was observed in both yeast and human breast cells following UV-irradiation indicating a conserved eukaryotic damage response. Moreover, P-CTD cleavage in breast epithelial cells was BRCA1-dependent since damage-induced P-CTD cleavage was only observed in the mutant BRCA1 cell line HCC1937 following ectopic expression of wild type BRCA1. Finally, BRCA1, SPT5 and hyperphosphorylated RPB1 form a complex that was rapidly degraded following MMS treatment in wild type but not BRCA1 mutant breast cells. These results extend the mechanistic links between BRCA1 and transcriptional consequences in response to DNA damage and suggest an important role for RNAPII P-CTD cleavage in BRCA1-mediated cancer suppression.
Chromosomal Proteins, Non-Histone
RNA Polymerase II
Transcriptional Elongation Factors
Published Version (Please cite this version)10.1371/journal.pone.0001448
Publication InfoBennett, Craig B; Westmoreland, Tammy J; Verrier, Carmel S; Blanchette, Carrie AB; Sabin, Tiffany L; Phatnani, Hemali P; ... Marks, Jeffrey R (2008). Yeast screens identify the RNA polymerase II CTD and SPT5 as relevant targets of BRCA1 interaction. PLoS One, 3(1). pp. e1448. 10.1371/journal.pone.0001448. Retrieved from https://hdl.handle.net/10161/4482.
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Professor of Biochemistry
My laboratory studies the mechanisms by which different activities in the cell nucleus are connected to the transcription machinery via interactions with the hyper-phosphorylated C-terminal repeat domain (PCTD) of elongating RNA polymerase II. Differential phosphorylation of the CTD, as the RNAP proceeds through successive stages of transcription, orchestrates sequential recruitment of factors to the transcriptase; this serves to coordinate RNA processing
Professor of Surgery
I have been engaged in basic and applied cancer research for over 28 years beginning with my post-doctoral fellowship under Arnold Levine at Princeton. Since being appointed to the faculty in the Department of Surgery at Duke, my primary interest has been towards understanding breast and ovarian cancer. I am a charter member of the NCI-Early Detection Research Network (EDRN) and have been an integral scientist in the breast and gynecologic collaborative group for 15 years including leading th
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