The RING finger of BRCA1 confers ubiquitin ligase activity that is markedly enhanced when complexed with another RING-containing protein, BARD1, and is required for the function of this tumor suppressor protein in protecting genomic integrity. Here, we report that co-expression of BRCA1-(1-639) and BARD1 in bacteria can assemble a potent ubiquitin ligase activity. Purified BRCA1-(1-639)⅐BARD1 stimulated the Ubc5c-mediated monoubiquitination of histone H2A/H2AX in vitro, suggesting a possible role for BRCA1⅐BARD1 in modifying chromatin structure. Moreover, the truncated BRCA1⅐BARD1 complex exhibited efficient autoubiquitination activity in vitro capable of assembling non-lysine 48-linked polyubiquitin chains on both BRCA1-(1-639) and BARD1. When co-expressed in cells by transient transfection, the recombinant BRCA1-(1-300)⅐BARD1 complex was found to be associated with polyubiquitin chains, suggesting that BRCA1-(1-300)⅐BARD1 was ubiquitinated in vivo as well. These results raise the possibility that BRCA1⅐BARD1 acts to assemble non-lysine 48-linked polyubiquitin chains that may serve as part of a signaling platform required for coordinating DNA repair-related events.When mutated, BRCA1 confers a genetic predisposition to breast and ovarian cancer. Germline mutations can be attributed to tumorigenesis in 45% of families with a history of breast cancer and 90% of families with both breast and ovarian cancer. The penetrance is such that female carriers have an estimated 80% lifetime risk of developing breast cancer (1).As a tumor suppressor, BRCA1 exerts a pleiotropic effect, playing a role in the maintenance of genomic integrity. To this end, several functions have been ascribed to BRCA1 including double strand DNA break repair, transcription-coupled DNA repair, transcriptional regulation, chromatin remodeling, and ubiquitin ligation (2). Despite implications in a diverse array of cellular pathways, the exact mechanism by which BRCA1 executes its function remains largely unknown.Within the first one hundred residues of BRCA1, which contains 20% of the biologically relevant mutations (3), lies a RING domain. RING fingers are "cross-brace" structures formed by conserved cysteine and histidine residues that coordinate two zinc ions (4). The RING finger motif has been well established to be able to mediate ubiquitin ligation, an activity intrinsic to a significant subset of the RING protein family. It does so by interacting with E2 1 ubiquitin-conjugating enzymes (5-7) and thus constitutes one of the two classes of E3 ubiquitin ligases, the other being the HECT domain containing E3 ligases (8). Traditionally, a RING E3 ubiquitin ligase interacts with both an E2, which has previously been charged with ubiquitin activated by an E1 ubiquitin-activating enzyme, and a substrate to catalyze the transfer of ubiquitin from the E2 to the substrate. However, some RING fingers have been operationally defined as having ubiquitin ligase activity without substrates. It is common to test RING fingers by simply assaying for the E1-and...

The mRNA polyadenylation factor CstF interacts with the BRCA1-associated protein BARD1, and this interaction represses the nuclear mRNA polyadenylation machinery in vitro. Given the suspected role of BRCA1/BARD1 in DNA repair, we tested whether inhibition of mRNA processing is linked to DNA damage. Strikingly, we found that 3' cleavage in extracts from cells treated with hydroxyurea or ultraviolet light was strongly, but transiently, inhibited. Although no changes were detected in CstF, BARD1, and BRCA1 protein levels, increased amounts of a CstF/BARD1/BRCA1 complex were detected. Supporting the physiological significance of these results, a previously identified tumor-associated germline mutation in BARD1 (Gln564His) reduced binding to CstF and abrogated inhibition of polyadenylation. Together these results indicate a link between mRNA 3' processing and DNA repair and tumor suppression.

Mammalian cells exhibit a complex response to DNA damage. The tumor suppressor BRCA1 and associated protein BARD1 are thought to play an important role in this response, and our previous work demonstrated that this includes transient inhibition of the pre-mRNA 3 processing machinery. Here we provide evidence that this inhibition involves proteasomal degradation of a component necessary for processing, RNA polymerase II (RNAP II). We further show that RNAP IIO, the elongating form of the enzyme, is a specific in vitro target of the BRCA1/BARD1 ubiquitin ligase activity. Significantly, siRNA-mediated knockdown of BRCA1 and BARD1 resulted in stabilization of RNAP II after DNA damage. In addition, inhibition of 3 cleavage induced by DNA damage was reverted in extracts of BRCA1-, BARD1-, or BRCA1/BARD1-depleted cells. We also describe corresponding changes in the nuclear localization and/or accumulation of these factors following DNA damage. Our results support a model in which a BRCA1/BARD1-containing complex functions to initiate degradation of stalled RNAP IIO, inhibiting the coupled transcription-RNA processing machinery and facilitating repair.

We previously showed that mRNA 3 0 end cleavage reaction in cell extracts is strongly but transiently inhibited under DNA-damaging conditions. The cleavage stimulation factor-50 (CstF-50) has a role in this response, providing a link between transcription-coupled RNA processing and DNA repair. In this study, we show that CstF-50 interacts with nuclear poly(A)-specific ribonuclease (PARN) using in vitro and in extracts of UV-exposed cells. The CstF-50/ PARN complex formation has a role in the inhibition of 3 0 cleavage and activation of deadenylation upon DNA damage. Extending these results, we found that the tumour suppressor BARD1, which is involved in the UV-induced inhibition of 3 0 cleavage, strongly activates deadenylation by PARN in the presence of CstF-50, and that CstF-50/ BARD1 can revert the cap-binding protein-80 (CBP80)-mediated inhibition of PARN activity. We also provide evidence that PARN along with the CstF/BARD1 complex participates in the regulation of endogenous transcripts under DNA-damaging conditions. We speculate that the interplay between polyadenylation, deadenylation and tumour-suppressor factors might prevent the expression of prematurely terminated messengers, contributing to control of gene expression under different cellular conditions.