Yeast Screens Identify the RNA Polymerase II CTD and SPT5 as Relevant Targets of BRCA1 Interaction
Figure 3
BRCA1-induced lethality involves plasmid loss and requires a functional BRCA1-BRCT domain.
(A) “Pullback” of WT yeast containing either the BRCA1 expression plasmid or empty vector control. To induce BRCA1, dilutions of plasmid bearing cells were plated into cooled liquid GAL agar containing medium with (+URA) or without (-URA) uracil selection for the plasmid marker (URA3) as previously described [54]. At the indicated times, BRCA1 expression was repressed by overlaying with GLU+URA or GLU-URA agar. For some data points, error bars (+/− 1 SD) are contained within the symbol. Enhanced lethality under selective conditions indicates plasmid loss is a significant component of BRCA1-induced lethality. (B) Deletion of SPT4 suppresses the physical loss of the BRCA1 expression plasmid. Stationary cultures of WT and spt4Δ strains containing the BRCA1 plasmid were “split” into twice the volume of GAL or GLU to express or repress BRCA1 respectively. Expression of BRCA1 in WT cells (in GAL) resulted in a time dependent increase in plasmid loss as determined by Southern analysis. Cells deleted for SPT4 retain the BRCA1 plasmid following BRCA1 expression. When BRCA1 was repressed, plasmid was physically retained in both WT and spt4Δ strains. (C) SPT4 deletion suppresses plasmid loss following BRCA1 expression. The viability of cells from the experiment described in Panel B was determined by plating aliquots at the indicated times onto YPD agar medium and genetically determining plasmid loss by replica plating the resulting colonies onto GLU-URA plates to detect the presence or absence of the plasmid URA3 marker (URA+). The relative survival of colony forming ability following repression (in GLU; left panel, two experiments) or induction of BRCA1 (in GAL; right panel, four experiments) in liquid media is shown. A significant loss of the plasmid marker can be observed in the WT but not in the spt4Δ strain following BRCA1 expression. No significant loss of the BRCA1 plasmid can be seen for either strain when the cells were grown in GLU. (D) Breast cancer associated mutations within the BRCT domain of BRCA1 suppress lethality in yeast. A single base pair was mutated within the BRCA1 expression plasmid (I1766S). The relative survival of colony forming ability on GAL vs GLU (GAL/GLU survival, four experiments) was determined as described for Fig. 1B. Complete suppression of BRCA1-induced lethality in yeast was also observed following deletion of the C-terminal 10 amino acids of BRCA1 that also disrupts BRCT domain function (data not shown).