Recruitment Of BRCA1 Research Articles

TPX2 serves as a novel target for expanding the utility of PARPi in pancreatic cancer through conferring synthetic lethality

BackgroundPARP inhibitors (PARPi) have been licensed for the maintenance therapy of patients with metastatic pancreatic cancer carrying pathogenic germline BRCA1/2 mutations. However, mutations in BRCA1/2 are notably rare in pancreatic...

DOT1L-mediated RAP80 methylation promotes BRCA1 recruitment to elicit DNA repair

Breast Cancer Type 1 Susceptibility Protein (BRCA1) is a tumor-suppressor protein that regulates various cellular pathways, including those that are essential for preserving genome stability. One essential mechanism involves a BRCA1-A complex that is recruited to double-strand breaks (DSBs) by RAP80 before initiating DNA damage repair (DDR). How RAP80 itself is recruited to DNA damage sites, however, is unclear. Here, we demonstrate an intrinsic correlation between a methyltransferase DOT1L-mediated RAP80 methylation and BRCA1-A complex chromatin recruitment that occurs during cancer cell radiotherapy resistance. Mechanistically, DOT1L is quickly recruited onto chromatin and methylates RAP80 at multiple lysines in response to DNA damage. Methylated RAP80 is then indispensable for binding to ubiquitinated H2A and subsequently triggering BRCA1-A complex recruitment onto DSBs. Importantly, DOT1L-catalyzed RAP80 methylation and recruitment of BRCA1 have clinical relevance, as inhibition of DOT1L or RAP80 methylation seems to enhance the radiosensitivity of cancer cells both in vivo and in vitro. These data reveal a crucial role for DOT1L in DDR through initiating recruitment of RAP80 and BRCA1 onto chromatin and underscore a therapeutic strategy based on targeting DOT1L to overcome tumor radiotherapy resistance.

Systematic investigation of BRCA1-A, -B, and -C complexes and their functions in DNA damage response and DNA repair.

BRCA1, a breast cancer susceptibility gene, has emerged as a central mediator that brings together multiple signaling complexes in response to DNA damage. The A, B, and C complexes of BRCA1, which are formed based on their phosphorylation-dependent interactions with the BRCA1-C-terminal domains, contribute to the roles of BRCA1 in DNA repair and cell cycle checkpoint control. However, their functions in DNA damage response remain to be fully appreciated. Specifically, there has been no systematic investigation of the roles of BRCA1-A, -B, and -C complexes in the regulation of BRCA1 localization and functions, in part because of cellular lethality associated with loss of CtIP protein, which is an essential component in BRCA1-C complex. To systematically investigate the functions of these complexes in DNA damage response, we depleted a key component in each of these complexes. We used the degradation tag system to inducibly deplete endogenous CtIP and obtained a series of RAP80/FANCJ/CtIP single-, double-, and triple-knockout cells. We showed that loss of BRCA1-B/FANCJ and BRCA1-C/CtIP, but not BRCA1-A/RAP80, resulted in reduced cell proliferation and increased sensitivity to DNA damage. BRCA1-C/CtIP and BRCA1-A/RAP80 were involved in BRCA1 recruitment to sites of DNA damage. However, BRCA1-A/RAP80 was not essential for damage-induced BRCA1 localization. Instead, RAP80/H2AX and CtIP have redundant roles in BRCA1 recruitment. Altogether, our systematic analysis uncovers functional differences between BRCA1-A, -B, and -C complexes and provides new insights into the roles of these BRCA1-associated protein complexes in DNA damage response and DNA repair.

Ubiquitin-induced RNF168 condensation promotes DNA double-strand break repair

Rapid accumulation of repair factors at DNA double-strand breaks (DSBs) is essential for DSB repair. Several factors involved in DSB repair have been found undergoing liquid-liquid phase separation (LLPS) at DSB sites to facilitate DNA repair. RNF168, a RING-type E3 ubiquitin ligase, catalyzes H2A.X ubiquitination for recruiting DNA repair factors. Yet, whether RNF168 undergoes LLPS at DSB sites remains unclear. Here, we identified K63-linked polyubiquitin-triggered RNF168 condensation which further promoted RNF168-mediated DSB repair. RNF168 formed liquid-like condensates upon irradiation in the nucleus while purified RNF168 protein also condensed in vitro. An intrinsically disordered region containing amino acids 460-550 was identified as the essential domain for RNF168 condensation. Interestingly, LLPS of RNF168 was significantly enhanced by K63-linked polyubiquitin chains, and LLPS largely enhanced the RNF168-mediated H2A.X ubiquitination, suggesting a positive feedback loop to facilitate RNF168 rapid accumulation and its catalytic activity. Functionally, LLPS deficiency of RNF168 resulted in delayed recruitment of 53BP1 and BRCA1 and subsequent impairment in DSB repair. Taken together, our finding demonstrates the pivotal effect of LLPS in RNF168-mediated DSB repair.

HDAC4 influences the DNA damage response and counteracts senescence by assembling with HDAC1/HDAC2 to control H2BK120 acetylation and homology-directed repair.

Access to DNA is the first level of control in regulating gene transcription, a control that is also critical for maintaining DNA integrity. Cellular senescence is characterized by profound transcriptional rearrangements and accumulation of DNA lesions. Here, we discovered an epigenetic complex between HDAC4 and HDAC1/HDAC2 that is involved in the erase of H2BK120 acetylation. The HDAC4/HDAC1/HDAC2 complex modulates the efficiency of DNA repair by homologous recombination, through dynamic deacetylation of H2BK120. Deficiency of HDAC4 leads to accumulation of H2BK120ac, impaired recruitment of BRCA1 and CtIP to the site of lesions, accumulation of damaged DNA and senescence. In senescent cells this complex is disassembled because of increased proteasomal degradation of HDAC4. Forced expression of HDAC4 during RAS-induced senescence reduces the genomic spread of γH2AX. It also affects H2BK120ac levels, which are increased in DNA-damaged regions that accumulate during RAS-induced senescence. In summary, degradation of HDAC4 during senescence causes the accumulation of damaged DNA and contributes to the activation of the transcriptional program controlled by super-enhancers that maintains senescence.

Abstract 7109: Fasn inhibition enhances the efficacy of chemotherapy by inhibiting DNA damage response in colorectal cancer

Abstract Altered lipid metabolism plays a significant role in progression of colorectal cancer, however, its role in chemotherapy resistance remains poorly understood. We previously identified FASN, the rate limiting enzyme in de novo lipogenesis, as an important regulator of colorectal cancer progression. However, TVB-3664, a potent and specific small molecule inhibitor of FASN, showed limited effect in reducing tumor burden in pre-clinical studies as a stand-alone therapy. Therefore, the goal of this study is to evaluate the combinatorial efficacy of TVB-3664 in improving the therapeutic indices of standard chemotherapy in colorectal cancer. Using a combination of colon cancer cell lines and patient derived organoids we showed that TVB-3664 potentiates chemotherapy drug-induced double-strand DNA breaks (DSBs) and apoptotic cell death by altering histone acetylation levels. In addition, TVB-3664 treatment blocked DNA damage response (DDR) by decreasing ATM expression and Chk1/2 phosphorylation. Mechanistically, inhibition of FASN prevents the activation of both DSB repair pathways, namely homologous recombination (HR) and classical non-homologous end joining (NHEJ), by blocking BRCA1 and 53BP1 recruitment to γH2AX foci, respectively. Moreover, we found that the inhibitory effect of TVB-3664 on DNA repair requires ACLY-dependent acetylation as genetic or pharmacological inhibition of ACLY attenuates TVB-3664-inducded DNA damage. Functionally, combining TVB-3664 with irinotecan synergistically decreased xenograft tumor growth and significantly delayed tumor relapse after withdrawal of therapy. Taken together, we identified a novel mechanism by which FASN inhibitors improve the efficacy of conventional chemotherapy by blocking DNA damage response to delay tumor recurrence and overcome a major challenge in treating colorectal cancer patients. Citation Format: Moumita Banerjee, Yekaterina Zaytseva, Sumati Hasani, Piotr Ryachahou, Tadahide Izumi, B. Mark Evers, Tianyan Gao. Fasn inhibition enhances the efficacy of chemotherapy by inhibiting DNA damage response in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7109.

Abstract 5608: The BRCA1-associated role of CDK9 in response to DNA damage is independent of its canonical role in transcription

Abstract The understanding of the biological role of BRCA1 in maintaining genomic integrity through homologous recombination (HR) repair has driven the development of targeted treatments with PARP inhibitors (PARPi) in BRCA1-linked tumors with promising results. Recently, cyclin-dependent kinase 9 (CDK9) inhibitors have emerged as therapeutic options to treat cancer. We have previously implicated CDK9 in genome integrity maintenance through HR repair by modulating BRCA1 recruitment to double-stranded break sites. CDK9 is a component of the positive transcription elongation (PTEF-b) complex and more recently was proposed by the hereditary breast case-control (BEACCON) study as a putative breast cancer (BC) predisposition gene in non-BRCA families. Differently from BRCA1-linked BCs, loss-of-function mutations in CDK9 were significantly correlated with ER-positive and Lobular BCs. CDK9 expression is also elevated in metastatic and recurrent ovarian cancer (OC) tissue and correlates with poor prognosis in OC patients. Here we characterize the BRCA1/BARD1/CDK9 complex using protein-protein interaction assays. Our data indicate that the CDK9/BARD1 interaction occurs indirectly through BRCA1. We also generated a separation-of-function mutation in CDK9 (mutCDK9) that disrupts its interaction with BRCA1 but retains its kinase activity and preserves its role in transcription. We rescued the mutCDK9/BRCA1 interaction using a complementary mutation in BRCA1 (cmutBRCA1), that retains BRCA1 role in cell viability. Further, we employed CRISPR-based genome editing to generate genetically defined haploid HAP1 cell lines that express mutCDK9 in various TP53BP1 and BRCA1 backgrounds. The mutCDK9 cells preserve global RNA transcription and the phosphorylation status of the RNA polymerase II. We observed that mutCDK9 cells are more sensitive to the PARPi, rucaparib when compared to wild-type cells in proficient and deficient TP53BP1 backgrounds. However, mutCDK9 expression did not affect the sensitivity of HAP1 cells expressing cmutBRCA1. Collectively, our data suggest that CDK9 acts in genome integrity maintenance by HR through its interaction with BRCA1 independently of its canonical role in transcription. Citation Format: Thales C. Nepomuceno, Marcelo A. Carvalho, Alvaro N. Monteiro. The BRCA1-associated role of CDK9 in response to DNA damage is independent of its canonical role in transcription [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5608.

RAP80 Phase Separation at DNA Double-Strand Break Promotes BRCA1 Recruitment and Tumor Radio-Resistance

RAP80 has been characterized as a component of the BRCA1-A complex and is responsible for the recruitment of BRCA1 to DNA double-strand breaks (DSBs). However, we and others found that the recruitment of RAP80 and BRCA1 are not absolutely temporally synchronized, indicating that other mechanisms, apart from physical interaction, may be implicated. Recently, we and other groups have reported that liquid-liquid phase separation (LLPS) is a pivotal mechanism underlying DNA repair factors condensation at DSBs and their function. In this study, we aim to disclose whether RAP80 undergoes LLPS at DSBs and whether it is required for BRCA1 recruitment. To verify RAP80 is an LLPS protein and its function in DNA damage response (DDR): (1) candidate-mEGFP fusion protein formed condensates in cells and showed fluorescence recovery after photobleaching (FRAP); (2) candidate protein was expressed in Escherichia coli and purified with GST; (3) intrinsically disordered region (IDR) of RAP80 were predicted and tested in cell and in vitro; (4) lentivirus were used to construct RAP80-Knock out (KO) and RAP80 re-expression cell lines; (5) length gradient K63 poly-ubiquitin chains were chemically synthesized and incubated with RAP80 protein in vitro; (6) BRCA1 and RAP80 location were determined through immunofluorescence; (7) RAP80 protein expression in tissue was determined by IHC staining. Thin layer scanning and 3D reconstruction of the RAP80-mEGFP-expressing cells under a fluorescence microscope showed that RAP80-mEGFP formed spherical condensates with fast FRAP. Observation of purified proteins revealed that GST-RAP80-mEGFP protein formed liquid-like droplets, presenting as a FRAP and the fusion event among adjacent droplets. PEG-8000 and Ficol-400 strengthened the formation of GST-RAP80-mEGFP droplets in vitro. Later, we used a previously developed optoIDR tool to verify that IDR1 (1-254aa) is critical for RAP80 LLPS. To investigate whether the interaction between RAP80 and K63 poly-ubiquitin chains could enhance the condensation of RAP80, we chemically synthesized K63 ubiquitin chains and incubated them with purified GST-RAP80-mCherry proteins. The results showed that supplementation of ubiquitin multipolymer (poly-ubiquitin) significantly induced the LLPS of RAP80, and the ability of RAP80 condensates formation potency was positively correlated with the length of the ubiquitin chain. Consistent with their LLPS capacity, RAP80-WT-mEGFP, RAP80-(IDR1+AIR)-mEGFP groups showed prominent BRCA1 foci, while RAP80-IDR1-mEGFP and RAP80-(SIM+UIM)-mEGFP groups showed delayed BRCA1 recruitment. In rectal cancer tissues, positive staining of the RAP80 protein was mainly observed in the nucleus of cancer cells and high RAP80 expression was correlated with a shorter overall survival time. RAP80 undergoes LLPS to form liquid-like condensates at DSB sites, which is important for BRCA1 recruitment and enhances tumor radio-resistance.

Ubiquitinated H2A.X-Induced RNF168 Condensation Promotes DNA Double-Strand Break Repair and Tumor Radioresistance

Ubiquitination of histone is an essential process involved in DNA damage response (DSB) serving as scaffolds for DNA repair proteins, but how these factors are recruited so quickly and regulated in a spatiotemporal manner remains poorly understood. Liquid-liquid phase separation (LLPS) has recently emerged as a mechanism for membraneless condensation driven by multivalent interactions. In this study, we aimed to investigate the LLPS potential of RNF168, an E3 ligase essential for DSB repair, and the mechanism underlying its-mediated tumor radio-resistance. The intrinsic disordered domain (IDR) of RNF168 was determined by the PONDR website. The LLPS properties were validated by droplet formation in vivo and in vitro. RNF168-mEGFP were expressed in Escherichia coli and purified with GST tag. The synthesized K63-linked ubiquitin chains were added to mimic the interactions between RNF168 and radiation-induced ubiquitinated-histone. Effects of RNF168 LLPS on downstream proteins were verified by immunofluorescence. RNF168-mEGFP recombinant protein formed liquid-like droplets in vivo and co-localized with γ-H2A.X foci after irradiation. The droplet's fluorescence recovered quickly after photobleaching, which could be abolished by 1,6-hexanediol treatment or ATP deprivation. Purified RNF168-mEGFP protein also condensed in vitro, and the size and number of droplets were related to protein concentration, salt concentration, pH, and temperature. Condensation of RNF168 was dependent on the IDR (323-459 amino acid), and more importantly, enhanced by synthesized K63-linked ubiquitin chains. LLPS of RNF168 was required for recruitment of RNF168 to DSB and RNF168-mediated γ-H2A.X ubiquitination. LLPS deficiency of RNF168 resulted in decreased recruitment of 53BP1, BRCA1, and RAP80 proteins, resulting in impaired DSB repair and genomic instability. Notably, higher expression of RNF168 was correlated with a poorer response to neoadjuvant radiochemotherapy in rectal cancer patients. Finally, RNF168 condensate-induced tumor radioresistance was further verified in the xenograft model. RNF168 undergoes LLPS at the DSB site, which is determined by both the IDR domain and the interaction with K63-linked ubiquitin chains. Radiation-induced RNF168 condensation accelerates the accumulation of RNF168 and promotes the recruitment of downstream effectors to DSB, resulting in enhanced DSB repair and tumor radioresistance.

RAP80 phase separation at DNA double-strand break promotes BRCA1 recruitment.

RAP80 has been characterized as a component of the BRCA1-A complex and is responsible for the recruitment of BRCA1 to DNA double-strand breaks (DSBs). However, we and others found that the recruitment of RAP80 and BRCA1 were not absolutely temporally synchronized, indicating that other mechanisms, apart from physical interaction, might be implicated. Recently, liquid-liquid phase separation (LLPS) has been characterized as a novel mechanism for the organization of key signaling molecules to drive their particular cellular functions. Here, we characterized that RAP80 LLPS at DSB was required for RAP80-mediated BRCA1 recruitment. Both cellular and in vitro experiments showed that RAP80 phase separated at DSB, which was ascribed to a highly disordered region (IDR) at its N-terminal. Meanwhile, the Lys63-linked poly-ubiquitin chains that quickly formed after DSBs occur, strongly enhanced RAP80 phase separation and were responsible for the induction of RAP80 condensation at the DSB site. Most importantly, abolishing the condensation of RAP80 significantly suppressed the formation of BRCA1 foci, encovering a pivotal role of RAP80 condensates in BRCA1 recruitment and radiosensitivity. Together, our study disclosed a new mechanism underlying RAP80-mediated BRCA1 recruitment, which provided new insight into the role of phase separation in DSB repair.

Oocytes can repair DNA damage during meiosis viaamicrotubule-dependent recruitment of CIP2A-MDC1-TOPBP1 complex from spindle pole to chromosomes.

Because DNA double-strand breaks (DSBs) greatly threaten genomic integrity, effective DNA damage sensing and repair are essential for cellular survival in all organisms. However, DSB repair mainly occurs during interphase and is repressed during mitosis. Here, we show that, unlike mitotic cells, oocytes can repair DSBs during meiosis I through microtubule-dependent chromosomal recruitment of the CIP2A-MDC1-TOPBP1 complex from spindle poles. After DSB induction, we observed spindle shrinkage and stabilization, as well as BRCA1 and 53BP1 recruitment to chromosomes and subsequent DSB repair during meiosis I. Moreover, p-MDC1 and p-TOPBP1 were recruited from spindle poles to chromosomes in a CIP2A-dependent manner. This pole-to-chromosome relocation of the CIP2A-MDC1-TOPBP1 complex was impaired not only by depolymerizing microtubules but also by depleting CENP-A or HEC1, indicating that the kinetochore/centromere serves as a structural hub for microtubule-dependent transport of the CIP2A-MDC1-TOPBP1 complex. Mechanistically, DSB-induced CIP2A-MDC1-TOPBP1 relocation is regulated by PLK1 but not by ATM activity. Our data provide new insights into the critical crosstalk between chromosomes and spindle microtubules in response to DNA damage to maintain genomic stability during oocyte meiosis.

CHAMP1-POGZ counteracts the inhibitory effect of 53BP1 on homologous recombination and affects PARP inhibitor resistance

DNA double-strand break (DSB) repair-pathway choice regulated by 53BP1 and BRCA1 contributes to genome stability. 53BP1 cooperates with the REV7-Shieldin complex and inhibits DNA end resection to block homologous recombination (HR) and affects the sensitivity to inhibitors for poly (ADP-ribose) polymerases (PARPs) in BRCA1-deficient cells. Here, we show that a REV7 binding protein, CHAMP1 (chromosome alignment-maintaining phosphoprotein 1), has an opposite function of REV7 in DSB repair and promotes HR through DNA end resection together with POGZ (POGO transposable element with ZNF domain). CHAMP1 was recruited to laser-micro-irradiation-induced DSB sites and promotes HR, but not NHEJ. CHAMP1 depletion suppressed the recruitment of BRCA1, but not the recruitment of 53BP1, suggesting that CHAMP1 regulates DSB repair pathway in favor of HR. Depletion of either CHAMP1 or POGZ impaired the recruitment of phosphorylated RPA2 and CtIP (CtBP-interacting protein) at DSB sites, implying that CHAMP1, in complex with POGZ, promotes DNA end resection for HR. Furthermore, loss of CHAMP1 and POGZ restored the sensitivity to a PARP inhibitor in cells depleted of 53BP1 together with BRCA1. These data suggest that CHAMP1and POGZ counteract the inhibitory effect of 53BP1 on HR by promoting DNA end resection and affect the resistance to PARP inhibitors.

ZMYM2 restricts 53BP1 at DNA double-strand breaks to favor BRCA1 loading and homologous recombination.

An inability to repair DNA double-strand breaks (DSBs) threatens genome integrity and can contribute to human diseases, including cancer. Mammalian cells repair DSBs mainly through homologous recombination (HR) and nonhomologous end-joining (NHEJ). The choice between these pathways is regulated by the interplay between 53BP1 and BRCA1, whereby BRCA1 excludes 53BP1 to promote HR and 53BP1 limits BRCA1 to facilitate NHEJ. Here, we identify the zinc-finger proteins (ZnF), ZMYM2 and ZMYM3, as antagonizers of 53BP1 recruitment that facilitate HR protein recruitment and function at DNA breaks. Mechanistically, we show that ZMYM2 recruitment to DSBs and suppression of break-associated 53BP1 requires the SUMO E3 ligase PIAS4, as well as SUMO binding by ZMYM2. Cells deficient for ZMYM2/3 display genome instability, PARP inhibitor and ionizing radiation sensitivity and reduced HR repair. Importantly, depletion of 53BP1 in ZMYM2/3-deficient cells rescues BRCA1 recruitment to and HR repair of DSBs, suggesting that ZMYM2 and ZMYM3 primarily function to restrict 53BP1 engagement at breaks to favor BRCA1 loading that functions to channel breaks to HR repair. Identification of DNA repair functions for these poorly characterized ZnF proteins may shed light on their unknown contributions to human diseases, where they have been reported to be highly dysregulated, including in several cancers.

DNA damage-induced sumoylation of Sp1 induces its interaction with RNF4 and degradation in S phase to remove 53BP1 from DSBs and permit HR

The factors involved in DNA damage recognition and repair are tightly regulated to ensure proper repair pathway choice. The mechanism(s) that determines the cell cycle-dependent turnover of these DNA damage repair factors remains unclear. Here, we show that Sp1, which regulates double-strand break (DSB) repair pathway choice through localization of 53BP1, is sumoylated at Lys16 following DNA damage; Sp1 sumoylation is required for its degradation and the removal of both Sp1 and 53BP1 from DSB sites. Induction of DNA DSBs induces Sp1 phosphorylation at DSBs by ATM, which is necessary for the subsequent sumoylation of Sp1. In addition to this damage-induced ATM-dependent phosphorylation and sumoylation, phosphorylation of Sp1 at Ser59 by Cyclin A/cdk2 upon entry into S phase is necessary for recognition, ubiquitination and degradation by the SUMO-targeted E3 ubiquitin ligase, RNF4. Eliminating Sp1 sumoylation by mutation of Sp1 at Lys16 (K16R) precluded removal of both Sp1 and 53BP1 from DSBs in S phase, resulting in decreased BRCA1 recruitment and defective homologous recombination (HR). Like BRCA1 deficient cells, cells expressing Sp1K16R are sensitive to PARP inhibition due to failure to degrade Sp1 and recruit BRCA1 resulting in defective HR that is rescued by knockdown of 53BP1. These results reveal the dynamic regulation of Sp1 and its role in the assembly and disassembly of DNA repair factors at DSBs.

Roles of the SUMO-related enzymes, PIAS1, PIAS4, and RNF4, in DNA double-strand break repair by homologous recombination

Post-translational modification of proteins by small ubiquitin-like modifier (SUMO) is known to be involved in a variety of cellular events. This modification, called SUMOylation, is carried out by the E1 activating enzyme, the E2 conjugating enzyme, and multiple E3 ligases. Previous studies have demonstrated that the SUMO E3 ligases, protein inhibitors of activated STAT 1 (PIAS1) and 4 (PIAS4), and the SUMO-targeted ubiquitin ligase, RING finger protein 4 (RNF4), play important roles in the repair of DNA double-strand breaks (DSBs). However, the mechanism by which these SUMO-related enzymes promote DSB repair is still poorly understood. In the present study, we focused on homologous recombination (HR), the most accurate DSB repair pathway, and aimed to elucidate the mechanism by which PIAS1, PIAS4, and RNF4 promote HR. In γ-ray-irradiated normal human fibroblasts, DSB end resection and RAD51 loading, the two essential steps of HR, were significantly impaired by small interfering RNA (siRNA)-mediated depletion of PIAS1, PIAS4, or RNF4. The recruitment of BRCA1, a major HR factor, to DSB sites was reduced in cells depleted of these SUMO-related enzymes. Consistent with the role of BRCA1 in counteracting the p53-binding protein 1 (53BP1)-mediated resection blockade, 53BP1 depletion rescued the reduced resection and RAD51 loading in the cells depleted of PIAS1, PIAS4, or RNF4. Moreover, Rap1-interacting factor 1 (RIF1), a resection inhibitor downstream of 53BP1, became more abundant at DSBs when PIAS1, PIAS4, RNF4, or BRCA1 was depleted. Importantly, the concomitant depletion of BRCA1 with either one of the SUMO-related enzymes did not further increase RIF1 at DSBs, when compared to single depletion of BRCA1. Collectively, these results suggest that PIAS1, PIAS4, RNF4, and BRCA1 work epistatically to counteract 53BP1/RIF1-mediated resection blockade, thereby promoting resection.

MYCN recruits the nuclear exosome complex to RNA polymerase II to prevent transcription-replication conflicts

The MYCN oncoprotein drives the development of numerous neuroendocrine and pediatric tumors. Here we show that MYCN interacts with the nuclear RNA exosome, a 3'-5' exoribonuclease complex, and recruits the exosome to its target genes. In the absence of the exosome, MYCN-directed elongation by RNA polymerase II (RNAPII) is slow and non-productive on a large group of cell-cycle-regulated genes. During the S phase of MYCN-driven tumor cells, the exosome is required to prevent the accumulation of stalled replication forks and of double-strand breaks close to the transcription start sites. Upon depletion of the exosome, activation of ATM causes recruitment of BRCA1, which stabilizes nuclear mRNA decapping complexes, leading to MYCN-dependent transcription termination. Disruption of mRNA decapping in turn activates ATR, indicating transcription-replication conflicts. We propose that exosome recruitment by MYCN maintains productive transcription elongation during S phase and prevents transcription-replication conflicts to maintain the rapid proliferation of neuroendocrine tumor cells.

SARS-CoV-2 Spike Impairs DNA Damage Repair and Inhibits V(D)J Recombination In Vitro.

Severe acute respiratory syndrome coronavirus 2 (SARS–CoV–2) has led to the coronavirus disease 2019 (COVID–19) pandemic, severely affecting public health and the global economy. Adaptive immunity plays a crucial role in fighting against SARS–CoV–2 infection and directly influences the clinical outcomes of patients. Clinical studies have indicated that patients with severe COVID–19 exhibit delayed and weak adaptive immune responses; however, the mechanism by which SARS–CoV–2 impedes adaptive immunity remains unclear. Here, by using an in vitro cell line, we report that the SARS–CoV–2 spike protein significantly inhibits DNA damage repair, which is required for effective V(D)J recombination in adaptive immunity. Mechanistically, we found that the spike protein localizes in the nucleus and inhibits DNA damage repair by impeding key DNA repair protein BRCA1 and 53BP1 recruitment to the damage site. Our findings reveal a potential molecular mechanism by which the spike protein might impede adaptive immunity and underscore the potential side effects of full-length spike-based vaccines.

BARD1 reads H2A lysine 15 ubiquitination to direct homologous recombination.

Protein ubiquitination at sites of DNA double-strand breaks (DSBs) by RNF168 recruits BRCA1 and 53BP11,2, which are mediators of the homologous recombination and non-homologous end joining DSB repair pathways, respectively3. Non-homologous end joining relies on 53BP1 binding directly to ubiquitinated lysine15 on H2A-type histones (H2AK15ub)4,5 (which is an RNF168-dependent modification6), but how RNF168 promotes BRCA1 recruitment and function remains unclear. Here we identify a tandem BRCT-domain-associated ubiquitin-dependent recruitment motif (BUDR) in BRCA1-associated RING domain protein1 (BARD1) (the obligate partner protein of BRCA1) that, by engaging H2AK15ub, recruits BRCA1 to DSBs. Disruption of the BUDR of BARD1 compromises homologous recombination and renders cells hypersensitive to PARP inhibition and cisplatin. We further show that BARD1 binds nucleosomes through multivalent interactions: coordinated binding of H2AK15ub and unmethylated H4 lysine20 by its adjacent BUDR and ankyrin repeat domains, respectively, provides high-affinity recognition of DNA lesions in replicated chromatin and promotes the homologous recombination activities of the BRCA1-BARD1 complex. Finally, our genetic epistasis experiments confirm that the need for BARD1 chromatin-binding activities can be entirely relieved upon deletion of RNF168 or 53BP1. Thus, our results demonstrate that by sensing DNA-damage-dependent and post-replication histone post-translation modification states, BRCA1-BARD1 complexes coordinate the antagonization of the 53BP1 pathway with promotion of homologous recombination, establishing a simple paradigm for the governance of the choice of DSB repair pathway.

DHX9-dependent recruitment of BRCA1 to RNA promotes DNA end resection in homologous recombination

Double stranded DNA Breaks (DSB) that occur in highly transcribed regions of the genome are preferentially repaired by homologous recombination repair (HR). However, the mechanisms that link transcription with HR are unknown. Here we identify a critical role for DHX9, a RNA helicase involved in the processing of pre-mRNA during transcription, in the initiation of HR. Cells that are deficient in DHX9 are impaired in the recruitment of RPA and RAD51 to sites of DNA damage and fail to repair DSB by HR. Consequently, these cells are hypersensitive to treatment with agents such as camptothecin and Olaparib that block transcription and generate DSB that specifically require HR for their repair. We show that DHX9 plays a critical role in HR by promoting the recruitment of BRCA1 to RNA as part of the RNA Polymerase II transcription complex, where it facilitates the resection of DSB. Moreover, defects in DHX9 also lead to impaired ATR-mediated damage signalling and an inability to restart DNA replication at camptothecin-induced DSB. Together, our data reveal a previously unknown role for DHX9 in the DNA Damage Response that provides a critical link between RNA, RNA Pol II and the repair of DNA damage by homologous recombination.

Super-resolution mapping of cellular double-strand break resection complexes during homologous recombination

Homologous recombination (HR) is a major pathway for repair of DNA double-strand breaks (DSBs). The initial step that drives the HR process is resection of DNA at the DSB, during which a multitude of nucleases, mediators, and signaling proteins accumulates at the damage foci in a manner that remains elusive. Using single-molecule localization super-resolution (SR) imaging assays, we specifically visualize the spatiotemporal behavior of key mediator and nuclease proteins as they resect DNA at single-ended double-strand breaks (seDSBs) formed at collapsed replication forks. By characterizing these associations, we reveal the in vivo dynamics of resection complexes involved in generating the long single-stranded DNA (ssDNA) overhang prior to homology search. We show that 53BP1, a protein known to antagonize HR, is recruited to seDSB foci during early resection but is spatially separated from repair activities. Contemporaneously, CtBP-interacting protein (CtIP) and MRN (MRE11-RAD51-NBS1) associate with seDSBs, interacting with each other and BRCA1. The HR nucleases EXO1 and DNA2 are also recruited and colocalize with each other and with the repair helicase Bloom syndrome protein (BLM), demonstrating multiple simultaneous resection events. Quantification of replication protein A (RPA) accumulation and ssDNA generation shows that resection is completed 2 to 4 h after break induction. However, both BRCA1 and BLM persist later into HR, demonstrating potential roles in homology search and repair resolution. Furthermore, we show that initial recruitment of BRCA1 and removal of Ku are largely independent of MRE11 exonuclease activity but dependent on MRE11 endonuclease activity. Combined, our observations provide a detailed description of resection during HR repair.