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Identification of a BRCA1-mRNA splicing complex required for efficient DNA repair and maintenance of genomic stability.

Savage KI, Gorski JJ, Barros EM, Irwin GW, Manti L, Powell AJ, Pellagatti A, Lukashchuk N, McCance DJ, McCluggage WG, Schettino G, Salto-Tellez M, Boultwood J, Richard DJ, McDade SS, Harkin DP - Mol. Cell (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Centre for Cancer Research and Cell Biology, Queen's University Belfast, 97 Lisburn Road, Belfast BT9 7BL, UK. Electronic address: k.savage@qub.ac.uk.

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BCLAF1 Interacts with Phosphorylated BRCA1 following DNA Damage(A) Colloidal Coomassie-stained gel of peptide pull-down assays carried out from 293T cell nuclear extracts with phosphorylated BRCA1-S1423 peptide and its nonphosphorylated counterpart. The indicated phosphopeptide-interacting band was identified as BCLAF1 by LC-MS/MS.(B) Coimmunoprecipitation assay demonstrating an interaction between BRCA1 and BCLAF1 in 293T cells treated with etoposide (1 μM, 16 hr), IR (2 Gy, 1 hr), MMS (200 μM, 6 hr), and HU (5 μM, 3 hr).(C) Coimmunoprecipitation assay demonstrating DNA damage-induced interaction of BCLAF1 with ectopic Flag-BRCA1 is abrogated by BRCA1-S1423A phosphosite substitution. A Flag-BRCA1 IP was also carried out from cells depleted of BCLAF1 to confirm the specificity of the BCLAF1 antibody.(D) Mapping of the BRCA1-interacting region within BCLAF1. Coimmunoprecipitation experiments were carried out from etoposide-treated cells transfected with the Flag-BCLAF1 truncation mutant constructs depicted in (E).(E) Schematic diagram of BCLAF1 truncation constructs used for BRCA1 coimmunoprecipitation experiments in (D).(F) Peptide pull-down assays carried out with [35S] in vitro-translated BCLAF1, indicating that BCLAF1 interacts directly and specifically with the phosphorylated BRCA1-S1423 peptide and not its unphosphorylated counterpart. See also Figure S1.
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fig1: BCLAF1 Interacts with Phosphorylated BRCA1 following DNA Damage(A) Colloidal Coomassie-stained gel of peptide pull-down assays carried out from 293T cell nuclear extracts with phosphorylated BRCA1-S1423 peptide and its nonphosphorylated counterpart. The indicated phosphopeptide-interacting band was identified as BCLAF1 by LC-MS/MS.(B) Coimmunoprecipitation assay demonstrating an interaction between BRCA1 and BCLAF1 in 293T cells treated with etoposide (1 μM, 16 hr), IR (2 Gy, 1 hr), MMS (200 μM, 6 hr), and HU (5 μM, 3 hr).(C) Coimmunoprecipitation assay demonstrating DNA damage-induced interaction of BCLAF1 with ectopic Flag-BRCA1 is abrogated by BRCA1-S1423A phosphosite substitution. A Flag-BRCA1 IP was also carried out from cells depleted of BCLAF1 to confirm the specificity of the BCLAF1 antibody.(D) Mapping of the BRCA1-interacting region within BCLAF1. Coimmunoprecipitation experiments were carried out from etoposide-treated cells transfected with the Flag-BCLAF1 truncation mutant constructs depicted in (E).(E) Schematic diagram of BCLAF1 truncation constructs used for BRCA1 coimmunoprecipitation experiments in (D).(F) Peptide pull-down assays carried out with [35S] in vitro-translated BCLAF1, indicating that BCLAF1 interacts directly and specifically with the phosphorylated BRCA1-S1423 peptide and not its unphosphorylated counterpart. See also Figure S1.

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).


Identification of a BRCA1-mRNA splicing complex required for efficient DNA repair and maintenance of genomic stability.

Savage KI, Gorski JJ, Barros EM, Irwin GW, Manti L, Powell AJ, Pellagatti A, Lukashchuk N, McCance DJ, McCluggage WG, Schettino G, Salto-Tellez M, Boultwood J, Richard DJ, McDade SS, Harkin DP - Mol. Cell (2014)

BCLAF1 Interacts with Phosphorylated BRCA1 following DNA Damage(A) Colloidal Coomassie-stained gel of peptide pull-down assays carried out from 293T cell nuclear extracts with phosphorylated BRCA1-S1423 peptide and its nonphosphorylated counterpart. The indicated phosphopeptide-interacting band was identified as BCLAF1 by LC-MS/MS.(B) Coimmunoprecipitation assay demonstrating an interaction between BRCA1 and BCLAF1 in 293T cells treated with etoposide (1 μM, 16 hr), IR (2 Gy, 1 hr), MMS (200 μM, 6 hr), and HU (5 μM, 3 hr).(C) Coimmunoprecipitation assay demonstrating DNA damage-induced interaction of BCLAF1 with ectopic Flag-BRCA1 is abrogated by BRCA1-S1423A phosphosite substitution. A Flag-BRCA1 IP was also carried out from cells depleted of BCLAF1 to confirm the specificity of the BCLAF1 antibody.(D) Mapping of the BRCA1-interacting region within BCLAF1. Coimmunoprecipitation experiments were carried out from etoposide-treated cells transfected with the Flag-BCLAF1 truncation mutant constructs depicted in (E).(E) Schematic diagram of BCLAF1 truncation constructs used for BRCA1 coimmunoprecipitation experiments in (D).(F) Peptide pull-down assays carried out with [35S] in vitro-translated BCLAF1, indicating that BCLAF1 interacts directly and specifically with the phosphorylated BRCA1-S1423 peptide and not its unphosphorylated counterpart. See also Figure S1.
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fig1: BCLAF1 Interacts with Phosphorylated BRCA1 following DNA Damage(A) Colloidal Coomassie-stained gel of peptide pull-down assays carried out from 293T cell nuclear extracts with phosphorylated BRCA1-S1423 peptide and its nonphosphorylated counterpart. The indicated phosphopeptide-interacting band was identified as BCLAF1 by LC-MS/MS.(B) Coimmunoprecipitation assay demonstrating an interaction between BRCA1 and BCLAF1 in 293T cells treated with etoposide (1 μM, 16 hr), IR (2 Gy, 1 hr), MMS (200 μM, 6 hr), and HU (5 μM, 3 hr).(C) Coimmunoprecipitation assay demonstrating DNA damage-induced interaction of BCLAF1 with ectopic Flag-BRCA1 is abrogated by BRCA1-S1423A phosphosite substitution. A Flag-BRCA1 IP was also carried out from cells depleted of BCLAF1 to confirm the specificity of the BCLAF1 antibody.(D) Mapping of the BRCA1-interacting region within BCLAF1. Coimmunoprecipitation experiments were carried out from etoposide-treated cells transfected with the Flag-BCLAF1 truncation mutant constructs depicted in (E).(E) Schematic diagram of BCLAF1 truncation constructs used for BRCA1 coimmunoprecipitation experiments in (D).(F) Peptide pull-down assays carried out with [35S] in vitro-translated BCLAF1, indicating that BCLAF1 interacts directly and specifically with the phosphorylated BRCA1-S1423 peptide and not its unphosphorylated counterpart. See also Figure S1.
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).

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.

View Article: PubMed Central - PubMed

Affiliation: Centre for Cancer Research and Cell Biology, Queen's University Belfast, 97 Lisburn Road, Belfast BT9 7BL, UK. Electronic address: k.savage@qub.ac.uk.

Show MeSH
Related in: MedlinePlus