BRCA1 Ubiquitinates RPB8 in Response to DNA Damage

Abstract

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 BRCA1-BARD1 mediates polyubiquitination of RPB8, a common subunit of RNA polymerases, in response to DNA damage. A proteomics screen identified RPB8 as a protein modified after epirubicin treatment in BRCA1-dependent manner. RPB8 interacted with BRCA1-BARD1 and was polyubiquitinated by BRCA1-BARD1 in vivo and in vitro. BRCA1-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 BRCA1-dependent cell survival after DNA damage. [Cancer Res 2007;67(3):951-8]

The UBXN1 Protein Associates with Autoubiquitinated Forms of the BRCA1 Tumor Suppressor and Inhibits Its Enzymatic Function

“…However, with certain combinations of other E2 enzymes, BRCA1/BARD1 can catalyze formation of either K48-or K63-linked polyubiquitin (7). To date, the only substrates shown to be ubiquitinated in a BRCA1-dependent manner in vivo are those conjugated to K6-linked chains, including NPM/B23, CtIP, RPB8, and BRCA1 itself (35,43,52,53,55). Interestingly, multiple lines of evidence suggest that proteins bearing K6-linked chains are not targeted for proteasomal 10).…”

“…Indeed, although BRCA1/BARD1 is reported to ubiquitinate a number of proteins in vitro (4,10,25,29,35,43,45,52,53,55), to date only four of these potential substrates (NPM, CtIP, RPB8, and BRCA1 itself) have been shown to be ubiquitinated in vivo in a BRCA1-dependent manner, and each was found to be conjugated to K6-linked chains (35,43,52,55). Interestingly, the steady-state levels of these substrates are not reduced by BRCA1/BARD1-dependent ubiquitination, suggesting that K6-linked chains do not promote proteasomal degradation in a manner analogous to that for K48-linked polyubiquitin (35,43,52,53,55). As such, the functional consequences of BRCA1-mediated ubiquitination with K6-linked chains remain unclear.…”

Human U2 snRNA Genes Exhibit a Persistently Open Transcriptional State and Promoter Disassembly at Metaphase

“…For example, if locus-specific fragility reflects occasional damage to U2 genes or occasional stalling of Pol II by secondary structure in the nascent U2 snRNA or DNA template (Fig. 6), loss of BRCA1 might cause fragility by impairing degradation or disassembly of the stalled transcription or repair complexes (46,106). Thus, the persistently open chromatin structure of the U2 snRNA genes may reflect a compromise between the benefits of rapid reinitiation and/or promoter clearance and the dangers of metaphase fragility.…”

Recruitment of Phosphorylated NPM1 to Sites of DNA Damage through RNF8-Dependent Ubiquitin Conjugates

“…Cells were cultured in DMEM as previously described (23)(24)(25). For IR, cells were exposed to X-ray irradiation (5 Gy) and cultured for the indicated time before analyses.…”