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

ABSTRACT: 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.

SUBMITTER: Sherker A

PROVIDER: S-EPMC8647010 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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, renders 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-SCDT-EMBOR-2021-53679V1 | biostudies-other

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

PARP-2 domain requirements for DNA damage-dependent activation and localization to sites of DNA damage.

Project description:Poly(ADP-ribose) polymerase-2 (PARP-2) is one of three human PARP enzymes that are potently activated during the cellular DNA damage response (DDR). DDR-PARPs detect DNA strand breaks, leading to a dramatic increase in their catalytic production of the posttranslational modification poly(ADP-ribose) (PAR) to facilitate repair. There are limited biochemical and structural insights into the functional domains of PARP-2, which has restricted our understanding of how PARP-2 is specialized toward specific repair pathways. PARP-2 has a modular architecture composed of a C-terminal catalytic domain (CAT), a central Trp-Gly-Arg (WGR) domain and an N-terminal region (NTR). Although the NTR is generally considered the key DNA-binding domain of PARP-2, we report here that all three domains of PARP-2 collectively contribute to interaction with DNA damage. Biophysical, structural and biochemical analyses indicate that the NTR is natively disordered, and is only required for activation on specific types of DNA damage. Interestingly, the NTR is not essential for PARP-2 localization to sites of DNA damage. Rather, the WGR and CAT domains function together to recruit PARP-2 to sites of DNA breaks. Our study differentiates the functions of PARP-2 domains from those of PARP-1, the other major DDR-PARP, and highlights the specialization of the multi-domain architectures of DDR-PARPs.

| S-EPMC4770219 | biostudies-literature

RNF4-dependent hybrid SUMO-ubiquitin chains are signals for RAP80 and thereby mediate the recruitment of BRCA1 to sites of DNA damage.

Project description:The DNA repair function of the breast cancer susceptibility protein BRCA1 depends in part on its interaction with RAP80, which targets BRCA1 to DNA double-strand breaks (DSBs) through recognition of K63-linked polyubiquitin chains. The localization of BRCA1 to DSBs also requires sumoylation. We demonstrated that, in addition to having ubiquitin-interacting motifs, RAP80 also contains a SUMO-interacting motif (SIM) that is critical for recruitment to DSBs. In combination with the ubiquitin-binding activity of RAP80, this SIM enabled RAP80 to bind with nanomolar affinity to hybrid chains consisting of ubiquitin conjugated to SUMO. Furthermore, RNF4, a SUMO-targeted ubiquitin E3 ligase that synthesizes hybrid SUMO-ubiquitin chains, localized to DSBs and was critical for the recruitment of RAP80 and BRCA1 to sites of DNA damage. Our findings, therefore, connect ubiquitin- and SUMO-dependent DSB recognition, revealing that RNF4-synthesized hybrid SUMO-ubiquitin chains are recognized by RAP80 to promote BRCA1 recruitment and DNA repair.

| S-EPMC4131685 | 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

Ring finger protein RNF169 antagonizes the ubiquitin-dependent signaling cascade at sites of DNA damage.

Project description:Ubiquitin signals emanating from DNA double-strand breaks (DSBs) trigger the ordered assembly of DNA damage mediator and repair proteins. This highly orchestrated process is accomplished, in part, through the concerted action of the RNF8 and RNF168 E3 ligases, which have emerged as core signaling intermediates that promote DSB-associated ubiquitylation events. In this study, we report the identification of RNF169 as a negative regulator of the DNA damage signaling cascade. We found that RNF169 interacted with ubiquitin structures and relocalized to DSBs in an RNF8/RNF168-dependent manner. Moreover, ectopic expression of RNF169 attenuated ubiquitin signaling and compromised 53BP1 accumulation at DNA damage sites, suggesting that RNF169 antagonizes RNF168 functions at DSBs. Our study unveils RNF169 as a component in DNA damage signal transduction and adds to the complexity of regulatory ubiquitylation in genome stability maintenance.

| S-EPMC3431699 | biostudies-literature

Histone ubiquitination associates with BRCA1-dependent DNA damage response.

Project description:Histone ubiquitination participates in multiple cellular processes, including the DNA damage response. However, the molecular mechanisms involved are not clear. Here, we have identified that RAP80/UIMC1 (ubiquitin interaction motif containing 1), a functional partner of BRCA1, recognizes ubiquitinated histones H2A and H2B. The interaction between RAP80 and ubiquitinated histones H2A and H2B is increased following DNA damage. Since RAP80 facilitates BRCA1's translocation to DNA damage sites, our results indicate that ubiquitinated histones H2A and H2B could be upstream partners of the BRCA1/RAP80 complex in the DNA damage response. Moreover, we have found that RNF8 (ring finger protein 8), an E3 ubiquitin ligase, regulates ubiquitination of both histones H2A and H2B. In RNF8-deficient mouse embryo fibroblasts, ubiquitination of both histones H2A and H2B is dramatically reduced, which abolishes the DNA damage-induced BRCA1 and RAP80 accumulation at damage lesions on the chromatin. Taken together, our results suggest that ubiquitinated histones H2A and H2B may recruit the BRCA1 complex to DNA damage lesions on the chromatin.

| S-EPMC2630672 | biostudies-literature

BRCA1 recruitment to damaged DNA sites is dependent on CDK9.

Project description:Double strand break lesions, the most toxic type of DNA damage, are repaired primarily through 2 distinct pathways: homology-directed recombination (HR) and non-homologous end-joining (NHEJ). BRCA1 and 53BP1, 2 proteins containing the BRCT modular domain, play an important role in DNA damage response (DDR) by orchestrating the decision between HR and NHEJ, but the precise mechanisms regarding both pathways are not entirely understood. Previously, our group identified a putative interaction between BRCA1 and BARD1 (BRCA1-associated RING domain 1) and the cyclin-dependent kinase (CDK9). CDK9 is a component of the positive transcription elongation complex and has been implicated in genome integrity maintenance associated with the replication stress response. Here we show that CDK9 interacts with endogenous BRCA1 and BARD1 mediated by their RING finger and BRCT domains, and describe CDK9 ionizing radiation-induced foci (IRIF) formation and its co-localization with BRCA1 in DNA damage sites. Cells lacking CDK9 are characterized by an altered ?-H2AX foci dynamics after DNA damage, a reduced efficiency in HR but not in NHEJ repair, failure to form BRCA1 and RAD51 IRIF and increased sensitivity to genotoxic agents. These data indicate that CDK9 is a player in the DDR and is consistent with its participation in HR pathway by modulating BRCA1 response.

| S-EPMC5397266 | biostudies-literature

p53-Dependent subcellular proteome localization following DNA damage.

Project description:The nucleolus is involved in regulating several aspects of stress responses and cell cycle arrest through the tumor suppressor p53. Under normal conditions, p53 is a short-lived protein that is present in cells at a barely detectable level. Upon exposure of cells to various forms of exogenous stress, such as DNA damage, there is a stabilization of p53 which is then responsible for an ensuing cascade of events. To further investigate the effect of p53 activation, we used a MS-based proteomics method to provide an unbiased, quantitative and high-throughput approach for measuring the subcellular distribution of the proteome that is dependent on p53. The spatial proteomics method analyses a whole cell extract created by recombining differentially labeled subcellular fractions derived from cells in which proteins have been mass labeled with heavy isotopes [Boisvert, F.-M., Lam, Y. W., Lamont, D., Lamond, A. I., Mol. Cell. Proteomics 2010, 9, 457-470]. This was used here to measure the relative distribution between cytoplasm, nucleus and nucleolus of around 2000 proteins in HCT116 cells that are either expressing wild-type p53 or null for p53. Spatial proteomics also facilitates a proteome-wide comparison of changes in protein localization in response to a wide range of physiological and experimental perturbations. We used this method to study differences in protein localization in HCT116 cells either with or without p53, and studied the differences in cellular response to DNA damage following treatment of HCT116 cells with etoposide in both p53 wild-type and null genetic backgrounds.

| S-EPMC3229981 | 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

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