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BGL3 lncRNA mediates retention of the BRCA1/BARD1 complex at DNA damage sites.

ABSTRACT: Long non-coding RNAs (lncRNAs) are emerging regulators of genomic stability and human disease. However, the molecular mechanisms by which nuclear lncRNAs directly contribute to DNA damage responses remain largely unknown. Using RNA antisense purification coupled with quantitative mass spectrometry (RAP-qMS), we found that the lncRNA BGL3 binds to PARP1 and BARD1, exhibiting unexpected roles in homologous recombination. Mechanistically, BGL3 is recruited to DNA double-strand breaks (DSBs) by PARP1 at an early time point, which requires its interaction with the DNA-binding domain of PARP1. BGL3 also binds the C-terminal BRCT domain and an internal region (amino acids 127-424) of BARD1, which mediates interaction of the BRCA1/BARD1 complex with its binding partners such as HP1γ and RAD51, resulting in BRCA1/BARD1 retention at DSBs. Cells depleted for BGL3 displayed genomic instability and were sensitive to DNA-damaging reagents. Overall, our findings underscore the biochemical versatility of RNA as a mediator molecule in the DNA damage response pathway, which affects the accumulation of BRCA1/BARD1 at DSBs.

SUBMITTER: Hu Z

PROVIDER: S-EPMC7298298 | biostudies-literature |

REPOSITORIES: biostudies-literature

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BGL3 lncRNA mediates retention of the BRCA1/BARD1 complex at DNA damage sites

Project description:Long non-coding RNAs (lncRNAs) are emerging regulators of genomic stability and human disease. However, the molecular mechanisms by which nuclear lncRNAs directly contribute to DNA damage responses remain largely unknown. Using RNA antisense purification coupled with quantitative mass spectrometry (RAP-qMS), we found that the lncRNA BGL3 binds to PARP1 and BARD1, exhibiting unexpected roles in homologous recombination. Mechanistically, BGL3 is recruited to DNA double-strand breaks (DSBs) by PARP1 at an early time point, which requires its interaction with the DNA-binding domain of PARP1. BGL3 also binds the C-terminal BRCT domain and an internal region (amino acids 127-424) of BARD1, which mediates interaction of the BRCA1/BARD1 complex with its binding partners such as HP1? and RAD51, resulting in BRCA1/BARD1 retention at DSBs. Cells depleted for BGL3 displayed genomic instability and were sensitive to DNA-damaging reagents. Overall, our findings underscore the biochemical versatility of RNA as a mediator molecule in the DNA damage response pathway, which affects the accumulation of BRCA1/BARD1 at DSBs.

| S-SCDT-EMBOJ-2019-104133 | biostudies-other

Interaction of BARD1 and HP1 Is Required for BRCA1 Retention at Sites of DNA Damage.

Project description:Stable retention of BRCA1/BARD1 complexes at sites of DNA damage is required for the proper response to DNA double-strand breaks (DSB). Here, we demonstrate that the BRCT domain of BARD1 is crucial for its retention through interaction with HP1. In response to DNA damage, BARD1 interacts with Lys9-dimethylated histone H3 (H3K9me2) in an ATM-dependent but RNF168-independent manner. This interaction is mediated primarily by HP1γ. A conserved HP1-binding motif in the BARD1 BRCT domain directly interacted with the chromoshadow domain of HP1 in vitro. Mutations in this motif (or simultaneous depletion of all three HP1 isoforms) disrupted retention of BARD1, BRCA1, and CtIP at DSB sites and allowed ectopic accumulation of RIF1, an effector of nonhomologous end-joining, at damaged loci in S-phase. UNC0638, a small-molecule inhibitor of histone lysine methyltransferase (HKMT), abolished retention and cooperated with the PARP inhibitor olaparib to block cancer cell growth. Taken together, our findings show how BARD1 promotes retention of the BRCA1/BARD1 complex at damaged DNA sites and suggest the use of HKMT inhibitors to leverage the application of PARP inhibitors to treat breast cancer.

| S-EPMC5003120 | biostudies-literature

RNF168-mediated localization of BARD1 recruits the BRCA1-PALB2 complex to DNA damage.

Project description:DNA damage prompts a diverse range of alterations to the chromatin landscape. The RNF168 E3 ubiquitin ligase catalyzes the mono-ubiquitination of histone H2A at lysine (K)13/15 (mUb-H2A), forming a binding module for DNA repair proteins. BRCA1 promotes homologous recombination (HR), in part, through its interaction with PALB2, and the formation of a larger BRCA1-PALB2-BRCA2-RAD51 (BRCA1-P) complex. The mechanism by which BRCA1-P is recruited to chromatin surrounding DNA breaks is unclear. In this study, we reveal that an RNF168-governed signaling pathway is responsible for localizing the BRCA1-P complex to DNA damage. Using mice harboring a Brca1CC (coiled coil) mutation that blocks the Brca1-Palb2 interaction, we uncovered an epistatic relationship between Rnf168- and Brca1CC alleles, which disrupted development, and reduced the efficiency of Palb2-Rad51 localization. Mechanistically, we show that RNF168-generated mUb-H2A recruits BARD1 through a BRCT domain ubiquitin-dependent recruitment motif (BUDR). Subsequently, BARD1-BRCA1 accumulate PALB2-RAD51 at DNA breaks via the CC domain-mediated BRCA1-PALB2 interaction. Together, these findings establish a series of molecular interactions that connect the DNA damage signaling and HR repair machinery.

| S-EPMC8373961 | biostudies-literature

Structure of BRCA1-BRCT/Abraxas Complex Reveals Phosphorylation-Dependent BRCT Dimerization at DNA Damage Sites.

Project description:BRCA1 accumulation at DNA damage sites is an important step for its function in the DNA damage response and in DNA repair. BRCA1-BRCT domains bind to proteins containing the phosphorylated serine-proline-x-phenylalanine (pSPxF) motif including Abraxas, Bach1/FancJ, and CtIP. In this study, we demonstrate that ionizing radiation (IR)-induces ATM-dependent phosphorylation of serine 404 (S404) next to the pSPxF motif. Crystal structures of BRCT/Abraxas show that phosphorylation of S404 is important for extensive interactions through the N-terminal sequence outside the pSPxF motif and leads to formation of a stable dimer. Mutation of S404 leads to deficiency in BRCA1 accumulation at DNA damage sites and cellular sensitivity to IR. In addition, two germline mutations of BRCA1 are found to disrupt the dimer interface and dimer formation. Thus, we demonstrate a mechanism involving IR-induced phosphorylation and dimerization of the BRCT/Abraxas complex for regulating Abraxas-mediated recruitment of BRCA1 in response to IR.

| S-EPMC4747905 | biostudies-literature

BRCA1/BARD1 complex associated RNA in HEK293T cells

Project description:We report the BRCA1/BARD1 complex associated RNA in HEK293T cells using RNA-seq following PAR-Clip.

2022-07-04 | GSE207338 | GEO

BRCA1-BARD1 promotes RAD51-mediated homologous DNA pairing.

Project description:The tumour suppressor complex BRCA1-BARD1 functions in the repair of DNA double-stranded breaks by homologous recombination. During this process, BRCA1-BARD1 facilitates the nucleolytic resection of DNA ends to generate a single-stranded template for the recruitment of another tumour suppressor complex, BRCA2-PALB2, and the recombinase RAD51. Here, by examining purified wild-type and mutant BRCA1-BARD1, we show that both BRCA1 and BARD1 bind DNA and interact with RAD51, and that BRCA1-BARD1 enhances the recombinase activity of RAD51. Mechanistically, BRCA1-BARD1 promotes the assembly of the synaptic complex, an essential intermediate in RAD51-mediated DNA joint formation. We provide evidence that BRCA1 and BARD1 are indispensable for RAD51 stimulation. Notably, BRCA1-BARD1 mutants with weakened RAD51 interactions show compromised DNA joint formation and impaired mediation of homologous recombination and DNA repair in cells. Our results identify a late role of BRCA1-BARD1 in homologous recombination, an attribute of the tumour suppressor complex that could be targeted in cancer therapy.

| S-EPMC5800781 | biostudies-literature

A conserved pathway to activate BRCA1-dependent ubiquitylation at DNA damage sites.

Project description:The BRCA1 tumour suppressor and its heterodimeric partner BARD1 constitute an E3-ubiquitin (Ub) ligase and function in DNA repair by unknown mechanisms. We show here that the Caenorhabditis elegans BRCA1/BARD1 (CeBCD) complex possesses an E3-Ub ligase responsible for ubiquitylation at DNA damage sites following ionizing radiation (IR). The DNA damage checkpoint promotes the association of the CeBCD complex with E2-Ub conjugating enzyme, Ubc5(LET-70), leading to the formation of an active E3-Ub ligase on chromatin following IR. Correspondingly, defects in Ubc5(let-70) or the DNA damage checkpoint genes atl-1 or mre-11 abolish CeBCD-dependent ubiquitylation in vivo. Extending these findings to human cells reveals a requirement for UbcH5c, the MRN complex, gamma-H2AX and a co-dependence for ATM and ATR kinases for BRCA1-dependent ubiquitylation at DNA damage sites. Furthermore, we demonstrate that the DNA damage checkpoint promotes the association between BRCA1 and UbcH5c to form an active E3-Ub ligase on chromatin after IR. These data reveal that BRCA1-dependent ubiquitylation is activated at sites of DNA repair by the checkpoint as part of a conserved DNA damage response.

| S-EPMC1462971 | biostudies-literature

Two redundant ubiquitin-dependent pathways of BRCA1 localization to DNA damage sites.

Project description:The tumor suppressor BRCA1 accumulates at sites of DNA damage in a ubiquitin-dependent manner. In this work, we revisit the role of RAP80 in promoting BRCA1 recruitment to damaged chromatin. We find that RAP80 acts redundantly with the BRCA1 RING domain to promote BRCA1 recruitment to DNA damage sites. We show that that RNF8 E3 ligase acts upstream of both the RAP80- and RING-dependent activities, whereas RNF168 acts uniquely upstream of the RING domain. BRCA1 RING mutations that do not impact BARD1 interaction, such as the E2 binding-deficient I26A mutation, render BRCA1 unable to accumulate at DNA damage sites in the absence of RAP80. Cells that combine BRCA1 I26A and mutations that disable the RAP80-BRCA1 interaction are hypersensitive to PARP inhibition and are unable to form RAD51 foci. Our results suggest that in the absence of RAP80, the BRCA1 E3 ligase activity is necessary for recognition of histone H2A Lys13/Lys15 ubiquitylation by BARD1, although we cannot rule out the possibility that the BRCA1 RING facilitates ubiquitylated nucleosome recognition in other ways.

| S-EPMC8647010 | biostudies-literature

USP13 regulates the RAP80-BRCA1 complex dependent DNA damage response.

Project description:BRCA1 regulates multiple cellular pathways that maintain genomic stability including cell cycle checkpoints, DNA repair, protein ubiquitination, chromatin remodelling, transcriptional regulation and apoptosis. Receptor-associated protein 80 (RAP80) helps recruit BRCA1 to double-strand breaks (DSBs) through the scaffold protein CCDC98 (Abraxas) and facilitates DNA damage response (DDR). However, the regulation of RAP80-BRCA1 complex is still unclear. Here we report that a deubiquitinase, USP13, regulates DDR by targeting RAP80. Mechanistically, USP13 is phosphorylated by ATM following DNA damage which, in turn, facilitates its DSB localization. USP13, in turn, deubiquitinates RAP80 and promotes RAP80 recruitment and proper DDR. Depleting or inhibiting USP13 sensitizes ovarian cancer cells to cisplatin and PARP inhibitor (olaparib) while overexpression of USP13 renders ovarian cancer cells resistant to chemotherapy. Overall, we identify USP13 as a regulator of DNA repair and reveal a model in which a phosphorylation-deubiquitination axis dynamically regulates RAP80-BRCA1 complex foci formation and function.

| S-EPMC5461494 | biostudies-literature

The deubiquitylating enzyme UCHL3 regulates Ku80 retention at sites of DNA damage.

Project description:Non-homologous end-joining (NHEJ), which can promote genomic instability when dysfunctional, is a major DNA double-strand break (DSB) repair pathway. Although ubiquitylation of the core NHEJ factor, Ku (Ku70-Ku80), which senses broken DNA ends, is important for its removal from sites of damage upon completion of NHEJ, the mechanism regulating Ku ubiquitylation remains elusive. We provide evidence showing that the ubiquitin carboxyl-terminal hydrolase L3 (UCHL3) interacts with and directly deubiquitylates one of the Ku heterodimer subunits, Ku80. Additionally, depleting UCHL3 resulted in reduced Ku80 foci formation, Ku80 binding to chromatin after DSB induction, moderately sensitized cells to ionizing radiation and decreased NHEJ efficiencies. Mechanistically, we show that DNA damage induces UCHL3 phosphorylation, which is dependent on ATM, downstream NHEJ factors and UCHL3 catalytic activity. Furthermore, this phosphorylation destabilizes UCHL3, despite having no effect on its catalytic activity. Collectively, these data suggest that UCHL3 facilitates cellular viability after DSB induction by antagonizing Ku80 ubiquitylation to enhance Ku80 retention at sites of damage.

| S-EPMC6297141 | biostudies-literature

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