Identification of a BRCA1-mRNA splicing complex required for efficient DNA repair and maintenance of genomic stability.
Bottom Line: Mutations within BRCA1 predispose carriers to a high risk of breast and ovarian cancers.Here, we report the identification of a DNA damage-induced BRCA1 protein complex containing BCLAF1 and other key components of the mRNA-splicing machinery.Interestingly, mutations in a number of proteins found within this complex have been identified in numerous cancer types.
Affiliation: Centre for Cancer Research and Cell Biology, Queen's University Belfast, 97 Lisburn Road, Belfast BT9 7BL, UK. Electronic address: email@example.com.Show MeSH
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Mentions: To examine how DNA damage-induced BRCA1 phosphorylation events might mechanistically regulate associated BRCA1 functions, we performed phosphopeptide pull-down assays followed by LC-MS/MS, to identify phospho-BRCA1-interacting proteins. Using this approach, we identified BCLAF1 as a BRCA1 phosphoserine-1423 (pSer-1423)-interacting protein (Figure 1A and see Figure S1A available online). Coimmunoprecipitation confirmed that BCLAF1 interacts with BRCA1 in response to DNA alkylation (MMS), stalled replication forks (HU), and DNA double-strand breaks (IR and etoposide) and is not mediated indirectly through RNA/DNA bridging (Figures 1B, S1B, and S1C). This suggests that this complex forms as part of a general DDR mechanism and is likely a reflection of BRCA1Ser-1423 being a substrate of both ATM and ATR, which are activated in response to DSBs or DNA single-strand breaks/stalled replication forks, respectively. In keeping with this, substitution of BRCA1Ser-1423 with alanine abrogated the damage-induced interaction between BRCA1 and BCLAF1, confirming BCLAF1 as a BRCA1pSer-1423 interacting protein (Figure 1C). Coimmunoprecipitation experiments with a series of Flag-tagged BCLAF1 truncated proteins (harvested from etoposide treated cells) revealed that deletion of the C-terminal region of BCLAF1 abolishes its ability to interact with BRCA1 (Figures 1D and 1E). The C terminus of BCLAF1 contains no defined domains, though it is positively charged under physiological conditions (pI = ∼9.5), suggesting that the interaction between BRCA1pSer-1423 and the BCLAF1 C terminus may occur directly. Consistent with this, in vitro-translated BCLAF1 bound strongly to the phosphorylated Ser1423-BRCA1 peptide, but not its nonphosphorylated counterpart (Figures 1F and S1D).
Affiliation: Centre for Cancer Research and Cell Biology, Queen's University Belfast, 97 Lisburn Road, Belfast BT9 7BL, UK. Electronic address: firstname.lastname@example.org.