E3 Ubiquitin Ligase RNF168 Research Articles

A RAD18-UBC13-PALB2-RNF168 axis mediates replication fork recovery in BRCA1-deficient cancer cells.

BRCA1/2 proteins function in genome stability by promoting repair of double-stranded DNA breaks through homologous recombination and by protecting stalled replication forks from nucleolytic degradation. In BRCA1/2-deficient cancer cells, extensively degraded replication forks can be rescued through distinct fork recovery mechanisms that also promote cell survival. Here, we identified a novel pathway mediated by the E3 ubiquitin ligase RAD18, the E2-conjugating enzyme UBC13, the recombination factor PALB2, the E3 ubiquitin ligase RNF168 and PCNA ubiquitination that promotes fork recovery in BRCA1- but not BRCA2-deficient cells. We show that this pathway does not promote fork recovery by preventing replication fork reversal and degradation in BRCA1-deficient cells. We propose a mechanism whereby the RAD18-UBC13-PALB2-RNF168 axis facilitates resumption of DNA synthesis by promoting re-annealing of the complementary single-stranded template strands of the extensively degraded forks, thereby allowing re-establishment of a functional replication fork. We also provide preliminary evidence for the potential clinical relevance of this novel fork recovery pathway in BRCA1-mutated cancers, as RAD18 is over-expressed in BRCA1-deficient cancers, and RAD18 loss compromises cell viability in BRCA1-deficient cancer cells.

Topology of ubiquitin chains in the e3 ubiquitin ligase rnf168 chromatosome entourage

Genome stability is critical for normal functioning of cells and depends on accuracy of DNA replication, chromosome segregation, and DNA repair. Cellular defense mechanisms against DNA damage are important for preventing the development of cancer and aging. The E3 ubiquitin ligase RNF168 of the RING superfamily is an essential component of the complex responsible for the ubiquitination of H2A/H2A.X histones near DNA double-strand breaks, which is a key step in attracting repair factors to the injury site. In this study, we unequivocally showed that RNF168 does not have ability to directly distinguish the architecture of polyubiquitin chains, except for tropism of its two ubiquitin-binding domains UDM1/2 to K63 ubiquitin chains. Analysis of the intracellular chromatosomal environment of full-length RNF168 and its domains by ligand-induced bioluminescence resonance energy transfer (BRET) revealed that the C-terminal part of UDM1 is associated with K63 ubiquitin chains; RING and the N-terminal part of UDM2 are sterically close to K63- and K48- ubiquitin chains, while the C-terminal part of UDM1 is colocalized with all possible ubiquitin variants. Our observations together with the available structural data suggest that the C-terminal part of UDM1 binds K63 polyubiquitin chains on linker histone H1; RING and the N-terminal part of UDM2 are located in the central part of the nucleosome and sterically close to H1 and K48-ubiquitinated alternative substrates of RNF168, such as JMJD2A/B demethylases, while the C-terminal part of UDM1 is in the region of an activated ubiquitin residue associated with E2 ubiquitin ligase, engaged by RNF168.

Topology of Ubiquitin Chains in the Chromatosomal Environment of the E3 Ubiquitin Ligase RNF168.

Genome stability is critical for normal functioning of cells, it depends on accuracy of DNA replication, chromosome segregation, and DNA repair. Cellular defense mechanisms against DNA damage are important for preventing cancer development and aging. The E3 ubiquitin ligase RNF168 of the RING superfamily is an essential component of the complex responsible for ubiquitination of the H2A/H2A.X histones near DNA double-strand breaks, which is a key step in attracting repair factors to the damage site. In this study, we unequivocally showed that RNF168 does not have the ability to directly distinguish architecture of polyubiquitin chains, except for the tropism of its two ubiquitin-binding domains UDM1/2 to K63 ubiquitin chains. Analysis of intracellular chromatosomal environment of the full-length RNF168 and its domains using the ligand-induced bioluminescence resonance energy transfer (BRET) revealed that the C-terminal part of UDM1 is associated with the K63 ubiquitin chains; RING and the N-terminal part of UDM2 are sterically close to the K63- and K48-ubiquitin chains, while the C-terminal part of UDM1 is co-localized with all possible ubiquitin variants. Our observations together with the available structural data suggest that the C-terminal part of UDM1 binds the K63 polyubiquitin chains on the linker histone H1; RING and the N-terminal part of UDM2 are located in the central part of nucleosome and sterically close to H1 and K48-ubiquitinated alternative substrates of RNF168, such as JMJD2A/B demethylases, while the C-terminal part of UDM1 is in the region of activated ubiquitin residue associated with E2 ubiquitin ligase, engaged by RNF168.

RNF168 suppresses the cancer stem cell-like traits of nonsmall cell lung cancer cells by mediating RhoC ubiquitination.

The critical roles of E3 ubiquitin ligase RNF168 have been widely revealed in various tumors, however, its roles in lung cancer progression are still confusing. Here, we found that RNF168 expression is positively correlated with the overall survival, first-progression survival, and postprogression survival of lung adenocarcinoma, but not correlated with these survivals of squamous cell carcinoma of lung. Furthermore, it was shown that RNF168 mRNA expression is lowly expressed in lung adenocarcinoma tissues, but highly expressed in squamous cell carcinoma of lung. Functional experiments indicated that RNF168 overexpression significantly suppressed the cancer stem cell (CSC)-like traits of nonsmall cell lung cancer (NSCLC) cells, as characterized by the attenuation of sphere-formation ability, ALDH activity, and the expression of lung CSC markers. Mechanistic studies demonstrated that RNF168 facilitated the ubiquitination of RhoC, which had been considered as a fascinating target for CSCs, and thus promoted RhoC protein degradation. Notably, RNF168 failed to affect the mRNA expression of RhoC and overexpression of RhoC rescued the inhibitory effects of RNF168 overexpression on the CSC-like traits of NSCLC cells. Therefore, this study revealed RNF168 as a novel regulator of RhoC protein in NSCLC cells, this RNF168/RhoC regulatory axis might be a potential target for NSCLC treatment.

The RNF8 and RNF168 Ubiquitin Ligases Regulate Pro- and Anti-Resection Activities at Broken DNA Ends During Non-Homologous End Joining

The RING-type E3 ubiquitin ligases RNF8 and RNF168 recruit DNA damage response (DDR) factors to chromatin flanking DNA double strand breaks (DSBs) including 53BP1, which protects DNA ends from resection during DNA DSB repair by non-homologous end joining (NHEJ). Deficiency of RNF8 or RNF168 does not lead to demonstrable NHEJ defects, but like deficiency of 53BP1, the combined deficiency of XLF and RNF8 or RNF168 leads to diminished NHEJ in lymphocytes arrested in G0/G1 phase. The function of RNF8 in NHEJ depends on its E3 ubiquitin ligase activity. Loss of RNF8 or RNF168 in G0/G1-phase lymphocytes leads to the resection of broken DNA ends, demonstrating that RNF8 and RNF168 function to protect DNA ends from nucleases, pos sibly through the recruitment of 53BP1. However, the loss of 53BP1 leads to more severe resection than the loss of RNF8 or RNF168. Moreover, in 53BP1-deficient cells, the loss of RNF8 or RNF168 leads to diminished DNA end resection. We conclude that RNF8 and RNF168 regulate pathways that both prevent and promote DNA end resection in cells arrested in G0/G1 phase.

ATM-Dependent Recruitment of BRD7 is required for Transcriptional Repression and DNA Repair at DNA Breaks Flanking Transcriptional Active Regions.

Repair of DNA double‐strand breaks (DSBs) is essential for genome integrity, and is accompanied by transcriptional repression at the DSB regions. However, the mechanisms how DNA repair induces transcriptional inhibition remain elusive. Here, it is identified that BRD7 participates in DNA damage response (DDR) and is recruited to the damaged chromatin via ATM signaling. Mechanistically, BRD7 joins the polycomb repressive complex 2 (PRC2), the nucleosome remodeling and histone deacetylation (NuRD) complex at the damaged DNA and recruits E3 ubiquitin ligase RNF168 to the DSBs. Furthermore, ATM‐mediated BRD7 phosphorylation is required for recruitment of the PRC2 complex, NuRD complex, DSB sensor complex MRE11‐RAD50‐NBS1 (MRN), and RNF168 to the active transcription sites at DSBs, resulting in transcriptional repression and DNA repair. Moreover, BRD7 deficiency sensitizes cancer cells to PARP inhibition. Collectively, BRD7 is crucial for DNA repair and DDR‐mediated transcription repression, which may serve as a therapeutic target. The findings identify the missing link between DNA repair and transcription regulation that maintains genome integrity.

Abstract 2377: Atm-dependent recruitment of brd7 is required for transcriptional repression and repair at dna breaks flanking transcriptional active regions

Abstract DNA double-strand break (DSB) is highly cytotoxic and deleterious lesions, causing gross chromosomal rearrangements, genomic instability and tumorigenesis (Meisenberg et al., 2019). In mammalian cells, such damages are mainly repaired through two pathways: non-homologous end joining (NHEJ) and homologous recombination (HR) (San Filippo et al., 2008). To avoid conflicts between ongoing transcription and repair activities, the DNA damage response (DDR) must coordinate the processes of repair signaling and local transcriptional capacity (Adam and Polo, 2014). Cells have evolved the ability of switching off transcription for DSB repair in ATM, DNA-PK and PARP1-dependent manner which is a critical step for maintaining genome-epigenome integrity (Iannelli et al., 2017; Shanbhag et al., 2010; Uckelmann and Sixma, 2017). The mono-ubiquitylation of H2A on K119 is one of the key determinants of transcriptional repression at sites of DNA damage (Kakarougkas et al., 2015; Rona et al., 2018; Shanbhag et al., 2010). However, depletion of these factors did not alleviate completely DSB-induced transcriptional repression, suggesting that additional yet unidentified factors or complexes are involved in the process. Here, we identified that BRD7, another specific subunit of PBAF complex, participated in DNA damage response (DDR) and was recruited to the damaged chromatin via ATM signaling. Mechanistically, BRD7 joined the polycomb repressive complex 2 (PRC2), the nucleosome remodeling and histone deacetylation (NuRD) complex at the damaged DNA and recruited E3 ubiquitin ligase RNF168 to the DSBs. Furthermore, ATM-mediated BRD7 phosphorylation is required for recruitment of the PRC2 complex, DSB sensor complex MRE11-RAD50-NBS1 (MRN) and RNF168 to the active transcription sites at DSBs, resulting in transcriptional repression and DNA repair. Moreover, BRD7 deficiency sensitized cancer cells to PARP inhibition. Collectively, BRD7 is crucial for DNA repair and DDR-mediated transcription repression, which may serve as a therapeutic target. Moreover, our findings have identified the missing link between DNA repair and transcription regulation that maintains genome integrity, as well as highlight the important role of PBAF complex in maintaining genomic integrity. Citation Format: Kaishun Hu. Atm-dependent recruitment of brd7 is required for transcriptional repression and repair at dna breaks flanking transcriptional active regions [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2377.

RNF8 has both KU-dependent and independent roles in chromosomal break repair.

Chromosomal double strand breaks (DSBs) can initiate several signaling events, such as ubiquitination, however the precise influence of such signaling on DSB repair outcomes remains poorly understood. With an RNA interference screen, we found that the E3 ubiquitin ligase RNF8 suppresses a deletion rearrangement mediated by canonical non-homologous end joining (C-NHEJ). We also found that RNF8 suppresses EJ without insertion/deletion mutations, which is a hallmark of C-NHEJ. Conversely, RNF8 promotes alternative EJ (ALT-EJ) events involving microhomology that is embedded from the edge of the DSB. These ALT-EJ events likely require limited end resection, whereas RNF8 is not required for single-strand annealing repair involving extensive end resection. Thus, RNF8 appears to specifically facilitate repair events requiring limited end resection, which we find is dependent on the DSB end protection factor KU. However, we also find that RNF8 is important for homology-directed repair (HDR) independently of KU, which appears linked to promoting PALB2 function. Finally, the influence of RNF8 on EJ is distinct from 53BP1 and the ALT-EJ factor, POLQ. We suggest that RNF8 mediates both ALT-EJ and HDR, but via distinct mechanisms, since only the former is dependent on KU.

Cadmium disrupts the DNA damage response by destabilizing RNF168

Cadmium (Cd) is a dispensable element for the human body and is usually considered a carcinogen. Occupational and environmental Cd exposure leads to sustained cellular proliferation in some tissues and tumorigenesis via an unclear mechanism. Here, we evaluated the role of Cd in the DNA damage response (DDR). We found that Cd exposure causes extensive DNA double-strand breaks (DSBs) and prevents accumulation of ubiquitination signals at these sites of DNA damage. Cd treatment compromises 53BP1 and BRCA1 recruitment to DSBs induced by itself or DNA damaging agents and partially inactivates the G2/M checkpoint. Mechanistically, Cd directly binds to the E3 ubiquitin ligase RNF168, induces the ubiquitin–proteasome pathway that mediates RNF168 degradation and suppresses RNF168 ubiquitin-ligase activity in vitro. Our study raises the possibility that Cd may target RNF168 to disrupt proper DSB signaling in cultured cells. This pathway may represent a novel mechanism for carcinogenesis induced by Cd.

Abstract 1754: Monoubiquitinated γ-H2AX - a more specific biomarker of DNA double-strand breaks

Abstract DNA double-strand brakes (DSBs) are a highly toxic form of DNA damage originating from many endogenous and exogenous sources. Cells employ specialized mechanisms for rapid detection and repair of these lesions. Histone H2AX phosphorylated at Ser139 (known as γ-H2AX) acts as a central platform for recruitment of other components of DSB repair. γ-H2AX assessment is currently the most widely used approach for quantitation of DSBs. However, γ-H2AX can be also induced by certain nongenotoxic stressors and during apoptosis, which can lead to false positive observations, especially in prolonged exposures. γ-H2AX undergoes RNF168-mediated K13/K15 monoubiquitination which is necessary for the recruitment of the downstream DSB repair factors. This ubiquitination event is rarely analyzed despite its functional importance. We examined formation and detection of γ-H2AX and its mono- (ub1) and diubiquitinated (ub2) forms in several human cell lines in response to mechanistically distinct inducers of DSBs and false-positive DSBs stressors. We found that γ-H2AX-ub forms were poorly detected using some common blotting procedures and antibodies. Under optimized conditions, γ-H2AX-ub1 was the predominant form accounting up to 80-90% of total γ-H2AX in primary human cells and its formation was strictly associated with the presence of DSBs. γ-H2AX and γ-H2AX-ub1 showed similar dose dependence and disappearance kinetics whereas H2AX-ub2 form was a more stable and dose-independent product. Unlike γ-H2AX, its ubiquitinated forms were not observed in the absence of DSBs (heat-shock or during apoptotic DNA cleavage). Apoptotic cells showed cleavage of the γ-H2AX-targeting E3 ubiquitin ligase RNF168. Our findings demonstrate that the ub1 form is a major fraction in the overall formation of γ-H2AX and its quantitation offers advantage in specificity for nonapoptotic DSBs. Citation Format: Michal W. Luczak, Anatoly Zhitkovich. Monoubiquitinated γ-H2AX - a more specific biomarker of DNA double-strand breaks [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1754.

RNF8 is associated with radioresistance, nodal stage and extranodal spread in oral cavity squamous-cell carcinoma

Background: Oral cavity cancer has an early metastatic profile and poor adjuvant therapy response when compared with other head and neck cancers, which is reflected in more aggressive clinical management. Accurate prognostic prediction is dependent on histological disease staging and extranodal spread, however these parameters are not available in preoperative clinical decision making. E3-ubiquitin ligase RNF8 is involved in the DNA repair cascade and dysregulation has been shown to promote in vitro genomic instability. Methods: A single-site retrospective series of 53 patients with oral cavity cancer were selected from 2006 to 2012. Clinical data was obtained from chart review, and survival data obtained from the Queensland Cancer Registry. Immunohistochemical analysis for RNF8 was performed against a constructed tissue microarray. Results: Sixteen (30.3%) tumours stained positive for RNF8 expression. RNF8 negative disease showed a significant survival advantage in patients who did not receive radiotherapy, while a non-significant inverse association was suggested in patients who did receive radiotherapy. RNF8 positivity was associated with increased tumour thickness (P=0.021), nodal extracapsular spread (ECS) (P=0.037) and pathological N-stage (P=0.036). Conclusions: RNF8 shows significant correlation with established tumour prognostic features of tumour thickness, ECS and nodal stage.

BRCA1 Haploinsufficiency Is Masked by RNF168-Mediated Chromatin Ubiquitylation

BRCA1 functions at two distinct steps during homologous recombination (HR). Initially, it promotes DNA end resection, and subsequently it recruits the PALB2 and BRCA2 mediator complex, which stabilizes RAD51-DNA nucleoprotein filaments. Loss of 53BP1 rescues the HR defect in BRCA1-deficient cells by increasing resection, suggesting that BRCA1's downstream role in RAD51 loading is dispensable when 53BP1 is absent. Here we show that the E3 ubiquitin ligase RNF168, in addition to its canonical role in inhibiting end resection, acts in a redundant manner with BRCA1 to load PALB2 onto damaged DNA. Loss of RNF168 negates the synthetic rescue of BRCA1 deficiency by 53BP1 deletion, and it predisposes BRCA1 heterozygous mice to cancer. BRCA1+/-RNF168-/- cells lack RAD51 foci and are hypersensitive to PARP inhibitor, whereas forced targeting of PALB2 to DNA breaks in mutant cells circumvents BRCA1 haploinsufficiency. Inhibiting the chromatin ubiquitin pathway may, therefore, be a synthetic lethality strategy for BRCA1-deficient cancers.

Ubiquitin ligase RNF8 suppresses Notch signaling to regulate mammary development and tumorigenesis.

The E3 ubiquitin ligase RNF8 plays critical roles in maintaining genomic stability by promoting the repair of DNA double-strand breaks (DSBs) through ubiquitin signaling. Abnormal activation of Notch signaling and defective repair of DSBs promote breast cancer risk. Here, we found that low expression of the full-length RNF8 correlated with poor prognosis for breast cancer patients. Our data revealed that in addition to its role in the repair of DSBs, RNF8 regulated Notch1 signaling and cell-fate determination of mammary luminal progenitors. Mechanistically, RNF8 acted as a negative regulator of Notch signaling by ubiquitylating the active NOTCH1 protein (N1ICD), leading to its degradation. Consistent with abnormal activation of Notch signaling and impaired repair of DSBs in Rnf8-mutant mammary epithelial cells, we observed increased risk of mammary tumorigenesis in mouse models for RNF8 deficiency. Notably, deficiency of RNF8 sensitized breast cancer cells to combination of pharmacological inhibitors of Notch signaling and poly(ADP-ribose) polymerase (PARP), suggesting implications for treatment of breast cancer associated with impaired RNF8 expression or function.

A20/TNFAIP3 Regulates the DNA Damage Response and Mediates Tumor Cell Resistance to DNA-Damaging Therapy.

A competent DNA damage response (DDR) helps prevent cancer, but once cancer has arisen, DDR can blunt the efficacy of chemotherapy and radiotherapy that cause lethal DNA breakage in cancer cells. Thus, blocking DDR may improve the efficacy of these modalities. Here, we report a new DDR mechanism that interfaces with inflammatory signaling and might be blocked to improve anticancer outcomes. Specifically, we report that the ubiquitin-editing enzyme A20/TNFAIP3 binds and inhibits the E3 ubiquitin ligase RNF168, which is responsible for regulating histone H2A turnover critical for proper DNA repair. A20 induced after DNA damage disrupted RNF168-H2A interaction in a manner independent of its enzymatic activity. Furthermore, it inhibited accumulation of RNF168 and downstream repair protein 53BP1 during DNA repair. A20 was also required for disassembly of RNF168 and 53BP1 from damage sites after repair. Conversely, A20 deletion increased the efficiency of error-prone nonhomologous DNA end-joining and decreased error-free DNA homologous recombination, destablizing the genome and increasing sensitivity to DNA damage. In clinical specimens of invasive breast carcinoma, A20 was widely overexpressed, consistent with its candidacy as a therapeutic target. Taken together, our findings suggest that A20 is critical for proper functioning of the DDR in cancer cells and it establishes a new link between this NFκB-regulated ubiquitin-editing enzyme and the DDR pathway.Significance: This study identifies the ubiquitin-editing enzyme A20 as a key factor in mediating cancer cell resistance to DNA-damaging therapy, with implications for blocking its function to leverage the efficacy of chemotherapy and radiotherapy. Cancer Res; 78(4); 1069-82. ©2017 AACR.

Retroviral insertional mutagenesis implicates E3 ubiquitin ligase RNF168 in the control of cell proliferation and survival.

The E3 ubiquitin ligase RNF168 is a ring finger protein that has been previously identified to play an important regulatory role in the repair of double-strand DNA breaks. In the present study, an unbiased forward genetics functional screen in mouse granulocyte/macrophage progenitor cell line FDCP1 has identified E3 ubiquitin ligase RNF168 as a key regulator of cell survival and proliferation. Our data indicate that RNF168 is an important component of the mechanisms controlling cell fate, not only in human and mouse haematopoietic growth factor dependent cells, but also in the human breast epithelial cell line MCF-7. These observations therefore suggest that RNF168 provides a connection to key pathways controlling cell fate, potentially through interaction with PML nuclear bodies and/or epigenetic control of gene expression. Our study is the first to demonstrate a critical role for RNF168 in the mechanisms regulating cell proliferation and survival, in addition to its well-established role in DNA repair.

Cell cycle-regulated ubiquitination of tankyrase 1 by RNF8 and ABRO1/BRCC36 controls the timing of sister telomere resolution.

Timely resolution of sister chromatid cohesion in G2/M is essential for genome integrity. Resolution at telomeres requires the poly(ADP-ribose) polymerase tankyrase 1, but the mechanism that times its action is unknown. Here, we show that tankyrase 1 activity at telomeres is controlled by a ubiquitination/deubiquitination cycle depending on opposing ubiquitin ligase and deubiquitinase activities. In late S/G2 phase, the DNA damage-responsive E3 ligase RNF8 conjugates K63-linked ubiquitin chains to tankyrase 1, while in G1 phase such ubiquitin chains are removed by BRISC, an ABRO1/BRCC36-containing deubiquitinase complex. We show that K63-linked ubiquitin chains accumulate on tankyrase 1 in late S/G2 to promote its stabilization, association with telomeres, and resolution of cohesion. Timing of this posttranslational modification coincides with the ATM-mediated DNA damage response that occurs on functional telomeres following replication in G2. Removal of ubiquitin chains is controlled by ABRO1/BRCC36 and occurs as cells exit mitosis and enter G1, ensuring that telomere cohesion is not resolved prematurely in S phase. Our studies suggest that a cell cycle-regulated posttranslational mechanism couples resolution of telomere cohesion with completion of telomere replication to ensure genome integrity.

The structural basis of modified nucleosome recognition by 53BP1.

DNA double-strand breaks (DSBs) elicit a histone modification cascade that controls DNA repair. This pathway involves the sequential ubiquitination of histones H1 and H2A by the E3 ubiquitin ligases RNF8 and RNF168, respectively. RNF168 ubiquitinates H2A on lysine 13 and lysine 15 (refs 7, 8) (yielding H2AK13ub and H2AK15ub, respectively), an event that triggers the recruitment of 53BP1 (also known as TP53BP1) to chromatin flanking DSBs. 53BP1 binds specifically to H2AK15ub-containing nucleosomes through a peptide segment termed the ubiquitination-dependent recruitment motif (UDR), which requires the simultaneous engagement of histone H4 lysine 20 dimethylation (H4K20me2) by its tandem Tudor domain. How 53BP1 interacts with these two histone marks in the nucleosomal context, how it recognizes ubiquitin, and how it discriminates between H2AK13ub and H2AK15ub is unknown. Here we present the electron cryomicroscopy (cryo-EM) structure of a dimerized human 53BP1 fragment bound to a H4K20me2-containing and H2AK15ub-containing nucleosome core particle (NCP-ubme) at 4.5 Å resolution. The structure reveals that H4K20me2 and H2AK15ub recognition involves intimate contacts with multiple nucleosomal elements including the acidic patch. Ubiquitin recognition by 53BP1 is unusual and involves the sandwiching of the UDR segment between ubiquitin and the NCP surface. The selectivity for H2AK15ub is imparted by two arginine fingers in the H2A amino-terminal tail, which straddle the nucleosomal DNA and serve to position ubiquitin over the NCP-bound UDR segment. The structure of the complex between NCP-ubme and 53BP1 reveals the basis of 53BP1 recruitment to DSB sites and illuminates how combinations of histone marks and nucleosomal elements cooperate to produce highly specific chromatin responses, such as those elicited following chromosome breaks.

Splicing controls the ubiquitin response during DNA double-strand break repair.

Although evidence that splicing regulates DNA repair is accumulating, the underlying mechanism(s) remain unclear. Here, we report that short-term inhibition of pre-mRNA splicing by spliceosomal inhibitors impairs cellular repair of DNA double-strand breaks. Indeed, interference with splicing as little as 1 h prior to irradiation reduced ubiquitylation of damaged chromatin and impaired recruitment of the repair factors WRAP53β, RNF168, 53BP1, BRCA1 and RAD51 to sites of DNA damage. Consequently, splicing-deficient cells exhibited significant numbers of residual γH2AX foci, as would be expected if DNA repair is defective. Furthermore, we show that this is due to downregulation of the E3 ubiquitin ligase RNF8 and that re-introduction of this protein into splicing-deficient cells restores ubiquitylation at sites of DNA damage, accumulation of downstream factors and subsequent repair. Moreover, downregulation of RNF8 explains the defective repair associated with knockdown of various splicing factors in recent genome-wide siRNA screens and, significantly, overexpression of RNF8 counteracts this defect. These discoveries reveal a mechanism that may not only explain how splicing regulates repair of double-strand breaks, but also may underlie various diseases caused by deregulation of splicing factors, including cancer.

RNF8 E3 Ubiquitin Ligase Stimulates Ubc13 E2 Conjugating Activity That Is Essential for DNA Double Strand Break Signaling and BRCA1 Tumor Suppressor Recruitment

DNA double strand break (DSB) responses depend on the sequential actions of the E3 ubiquitin ligases RNF8 and RNF168 plus E2 ubiquitin-conjugating enzyme Ubc13 to specifically generate histone Lys-63-linked ubiquitin chains in DSB signaling. Here, we defined the activated RNF8-Ubc13∼ubiquitin complex by x-ray crystallography and its functional solution conformations by x-ray scattering, as tested by separation-of-function mutations imaged in cells by immunofluorescence. The collective results show that the RING E3 RNF8 targets E2 Ubc13 to DSB sites and plays a critical role in damage signaling by stimulating polyubiquitination through modulating conformations of ubiquitin covalently linked to the Ubc13 active site. Structure-guided separation-of-function mutations show that the RNF8 E2 stimulating activity is essential for DSB signaling in mammalian cells and is necessary for downstream recruitment of 53BP1 and BRCA1. Chromatin-targeted RNF168 rescues 53BP1 recruitment involved in non-homologous end joining but not BRCA1 recruitment for homologous recombination. These findings suggest an allosteric approach to targeting the ubiquitin-docking cleft at the E2-E3 interface for possible interventions in cancer and chronic inflammation, and moreover, they establish an independent RNF8 role in BRCA1 recruitment.

Chronic oxidative stress promotes H2AX protein degradation and enhances chemosensitivity in breast cancer patients.

Anti‐cancer drugs often increase reactive oxygen species (ROS) and cause DNA damage. Here, we highlight a new cross talk between chronic oxidative stress and the histone variant H2AX, a key player in DNA repair. We observe that persistent accumulation of ROS, due to a deficient JunD‐/Nrf2‐antioxidant response, reduces H2AX protein levels. This effect is mediated by an enhanced interaction of H2AX with the E3 ubiquitin ligase RNF168, which is associated with H2AX poly‐ubiquitination and promotes its degradation by the proteasome. ROS‐mediated H2AX decrease plays a crucial role in chemosensitivity. Indeed, cycles of chemotherapy that sustainably increase ROS reduce H2AX protein levels in Triple‐Negative breast cancer (TNBC) patients. H2AX decrease by such treatment is associated with an impaired NRF2‐antioxidant response and is indicative of the therapeutic efficiency and survival of TNBC patients. Thus, our data describe a novel ROS‐mediated regulation of H2AX turnover, which provides new insights into genetic instability and treatment efficacy in TNBC patients.