BRCA1 ubiquitinates RPB8 in response to DNA damage

The breast and ovarian tumor suppressor BRCA1 catalyzes untraditional polyubiquitin chains that could be a signal for processes other than proteolysis. However, despite intense investigations, the mechanisms regulated by the enzyme activity remain only partially understood. Here, we report that BRCAl-BARDl mediates polyubiquitination of RPBB, a common subunit of RNA polymerases, in response to DNA damage. A proteomics screen identified RPBB as a protein modified after epirubicin treatment in BRCA1-dependent manner. RPB8 interacted with BRCAl-BARD1 and was polyubiquitinated by BRCAl-BARDl in vivo and in vitro. BRCAl-BARD1 did not destabilize RPB8 in vivo but rather caused an increase in the amount of soluble RPB8. Importantly, RPB8 was polyubiquitinated immediately after UV irradiation in a manner sensitive to BRCA1 knockdown by RNA interference. Substitution of five lysine residues of RPB8 with arginine residues abolished its ability to be ubiquitinated while preserving its polymerase activity. HeLa cell lines stably expressing this ubiquitin-resistant form of RPB8 exhibited UV hypersensitivity accompanied by up-regulated caspase activity. Our findings suggest that ubiquitination of a common subunit of RNA polymerases is a mechanism underlying BRCAl-dependent cell survival after DNA damage.