BCL10 regulates RNF8/RNF168-mediated ubiquitination in the DNA damage response

Timely and proper cellular response to DNA damage is essential for maintenance of genome stability and integrity. B-cell lymphoma/leukemia 10 (BCL10) facilitates ubiquitination of NEMO in the cytosol, activating NFκB signaling. Translocation and/or point mutations of BCL10 associate with mucosa-associated lymphoid tissue lymphomas and other malignancies. However, the mechanisms by which the resulting aberrant expression of BCL10 leads to cellular oncogenesis are poorly understood. In this report, we found that BCL10 in the nucleus is enriched at the DNA damage sites in an ATM- and RNF8-dependent manner. ATM-dependent phosphorylation of BCL10 promotes its interaction with and presentation of UBC13 to RNF8, and RNF8-mediated ubiquitination of BCL10 enhances binding of BCL10 and UBC13 to RNF168. This allows mono-ubiquitination on H2AX by RNF168 and further poly-ubiquitination by the RNF8/RNF168-containing complex. Depletion of BCL10 compromised homology recombination-mediated DNA double-strand break (DSB) repair because of insufficient recruitment of BRCA1, RAD51, and the ubiquitinated DNA damage response factors. Taken together, our results demonstrate a novel function of BCL10 in delivering UBC13 to RNF8/RNF168 to regulate ubiquitination-mediated DSB signaling and repair.     

Nucleotide excision repair–induced H2A ubiquitination is dependent on MDC1 and RNF8 and reveals a universal DNA damage response

Chromatin modifications are an important component of the of DNA damage response (DDR) network that safeguard genomic integrity. Recently, we demonstrated nucleotide excision repair (NER)–dependent histone H2A ubiquitination at sites of ultraviolet (UV)-induced DNA damage. In this study, we show a sustained H2A ubiquitination at damaged DNA, which requires dynamic ubiquitination by Ubc13 and RNF8. Depletion of these enzymes causes UV hypersensitivity without affecting NER, which is indicative of a function for Ubc13 and RNF8 in the downstream UV–DDR. RNF8 is targeted to damaged DNA through an interaction with the double-strand break (DSB)–DDR scaffold protein MDC1, establishing a novel function for MDC1. RNF8 is recruited to sites of UV damage in a cell cycle–independent fashion that requires NER-generated, single-stranded repair intermediates and ataxia telangiectasia–mutated and Rad3-related protein. Our results reveal a conserved pathway of DNA damage–induced H2A ubiquitination for both DSBs and UV lesions, including the recruitment of 53BP1 and Brca1. Although both lesions are processed by independent repair pathways and trigger signaling responses by distinct kinases, they eventually generate the same epigenetic mark, possibly functioning in DNA damage signal amplification.     

The RING Finger Protein RNF8 Ubiquitinates Nbs1 to Promote DNA Double-strand Break Repair by Homologous Recombination

Ubiquitination plays an important role in the DNA damage response. We identified a novel interaction of the E3 ubiquitin ligase RNF8 with Nbs1, a key regulator of DNA double-strand break (DSB) repair. We found that Nbs1 is ubiquitinated both before and after DNA damage and is a direct ubiquitination substrate of RNF8. We also identified key residues on Nbs1 that are ubiquitinated by RNF8. By using laser microirradiation and live-cell imaging, we observed that RNF8 and its ubiquitination activity are important for promoting optimal binding of Nbs1 to DSB-containing chromatin. We also demonstrated that RNF8-mediated ubiquitination of Nbs1 contributes to the efficient and stable binding of Nbs1 to DSBs and is important for HR-mediated DSB repair. Taken together, these studies suggest that Nbs1 is one important target of RNF8 to regulate DNA DSB repair.     

ATDC (Ataxia Telangiectasia Group D Complementing) Promotes Radioresistance through an Interaction with the RNF8 Ubiquitin Ligase

Induction of DNA damage by ionizing radiation (IR) and/or cytotoxic chemotherapy is an essential component of cancer therapy. The ataxia telangiectasia group D complementing gene (ATDC, also called TRIM29) is highly expressed in many malignancies. It participates in the DNA damage response downstream of ataxia telangiectasia-mutated (ATM) and p38/MK2 and promotes cell survival after IR. To elucidate the downstream mechanisms of ATDC-induced IR protection, we performed a mass spectrometry screen to identify ATDC binding partners. We identified a direct physical interaction between ATDC and the E3 ubiquitin ligase and DNA damage response protein, RNF8, which is required for ATDC-induced radioresistance. This interaction was refined to the C-terminal portion (amino acids 348–588) of ATDC and the RING domain of RNF8 and was disrupted by mutation of ATDC Ser-550 to alanine. Mutations disrupting this interaction abrogated ATDC-induced radioresistance. The interaction between RNF8 and ATDC, which was increased by IR, also promoted downstream DNA damage responses such as IR-induced γ-H2AX ubiquitination, 53BP1 phosphorylation, and subsequent resolution of the DNA damage foci. These studies define a novel function for ATDC in the RNF8-mediated DNA damage response and implicate RNF8 binding as a key determinant of the radioprotective function of ATDC.     

Significance of the melanocortin 1 receptor in the DNA damage response of human melanocytes to ultraviolet radiation

Activation of the melanocortin 1 receptor (MC1R) by α‐melanocortin (α‐MSH) stimulates eumelanin synthesis and enhances repair of ultraviolet radiation (UV)‐induced DNA damage. We report on the DNA damage response (DDR) of human melanocytes to UV and its enhancement by α‐MSH. α‐MSH up‐regulated the levels of XPC, the enzyme that recognizes DNA damage sites, enhanced the UV‐induced phosphorylation of the DNA damage sensors ataxia telangiectasia and Rad3‐related (ATR) and ataxia telangiectasia mutated (ATM) and their respect‐ive substrates checkpoint kinases 1 and 2, and increased phosphorylated H2AX (γH2AX) formation. These effects required functional MC1R and were absent in melanocytes expressing loss of function (LOF) MC1R. The levels of wild‐type p53‐induced phosphatase 1 (Wip1), which dephosphorylates γH2AX, correlated inversely with γH2AX. We propose that α‐MSH increases UV‐induced γH2AX to facilitate formation of DNA repair complexes and repair of DNA photoproducts, and LOF of MC1R compromises the DDR and genomic stability of melanocytes.     

Homozygous deficiency of ubiquitin-ligase ring-finger protein RNF168 mimics the radiosensitivity syndrome of ataxia-telangiectasia

Maintaining genomic integrity is critical to avoid life-threatening disorders, such as premature aging, neurodegeneration and cancer. A multiprotein cascade operates at sites of DNA double-strand breaks (DSBs) to recognize, signal and repair damage. RNF168 (ring-finger nuclear factor) contributes to this emerging pathway of several E3 ubiquitin ligases that perform sequential ubiquitylations on damaged chromosomes, chromatin modifications essential for aggregation of repair complexes at the DSB sites. Here, we report the clinical and cellular phenotypes associated with a newly identified homozygous nonsense mutation in the RNF168 gene of a patient with a syndrome mimicking ataxia-telangiectasia. The mutation eliminated both of RNF168's ubiquitin-binding motifs, thus blocking progression of the ubiquitylation cascade and retention of repair proteins including tumor suppressors 53BP1 and BRCA1 at DSB sites, consistent with the observed defective DNA damage checkpoints/repair and pronounced radiosensitivity. Rapid screening for RNF168 pathway deficiency was achieved by scoring patients' lymphoblastoid cells for irradiation-induced nuclear foci containing 53BP1, a robust assay we propose for future diagnostic applications. The formation of radiation-induced DSB repair foci was rescued by ectopic expression of wild-type RNF168 in patient's cells, further causally linking the RNF168 mutation with the pathology. Clinically, this novel syndrome featured ataxia, telangiectasia, elevated alphafetoprotein, immunodeficiency, microcephaly and pulmonary failure and has implications for the differential diagnosis of autosomal recessive ataxias.     

Critical role of monoubiquitination of histone H2AX protein in histone H2AX phosphorylation and DNA damage response

DNA damage response is an important surveillance mechanism used to maintain the integrity of the human genome in response to genotoxic stress. Histone variant H2AX is a critical sensor that undergoes phosphorylation at serine 139 upon genotoxic stress, which provides a docking site to recruit the mediator of DNA damage checkpoint protein 1 (MDC1) and DNA repair protein complex to sites of DNA breaks for DNA repair. Here, we show that monoubiquitination of H2AX is induced upon DNA double strand breaks and plays a critical role in H2AX Ser-139 phosphorylation (γ-H2AX), in turn facilitating the recruitment of MDC1 to DNA damage foci. Mechanistically, we show that monoubiquitination of H2AX induced by RING finger protein 2 (RNF2) is required for the recruitment of active ataxia telangiectasia mutated to DNA damage foci, thus affecting the formation of γ-H2AX. Importantly, a defect in monoubiquitination of H2AX profoundly enhances ionizing radiation sensitivity. Our study therefore suggests that monoubiquitination of H2AX is an important step for DNA damage response and may have important clinical implications for the treatment of cancers.     

CDK2 phosphorylation of Werner protein (WRN) contributes to WRN’s DNA double‐strand break repair pathway choice

Werner syndrome (WS) is an accelerated aging disorder characterized by genomic instability, which is caused by WRN protein deficiency. WRN participates in DNA metabolism including DNA repair. In a previous report, we showed that WRN protein is recruited to laser‐induced DNA double‐strand break (DSB) sites during various stages of the cell cycle with similar intensities, supporting that WRN participates in both non‐homologous end joining (NHEJ) and homologous recombination (HR). Here, we demonstrate that the phosphorylation of WRN by CDK2 on serine residue 426 is critical for WRN to make its DSB repair pathway choice between NHEJ and HR. Cells expressing WRN engineered to mimic the unphosphorylated or phosphorylation state at serine 426 showed abnormal DSB recruitment, altered RPA interaction, strand annealing, and DSB repair activities. The CDK2 phosphorylation on serine 426 stabilizes WRN’s affinity for RPA, likely increasing its long‐range resection at the end of DNA strands, which is a crucial step for HR. Collectively, the data shown here demonstrate that a CDK2‐dependent phosphorylation of WRN regulates DSB repair pathway choice and cell cycle participation.     

The BRCT domain and the specific loop 1 of human Polμ are targets of Cdk2/cyclin A phosphorylation

Human family X polymerases contribute both to genomic stability and variability through their specialized functions in DNA repair. Polμ participates in the repair of spontaneous double strand breaks (DSB) by non homologous end-joining (NHEJ), and also in the V(D)J recombination process after programmed DSBs. Polμ plays this dual role due to its template-dependent and terminal transferase (template-independent) polymerization activities. In this study we evaluated if Polμ could be regulated by Cdk phosphorylation along the cell cycle. In vitro kinase assays showed that the S phase-associated Cdk2/cyclin A complex was able to phosphorylate Polμ. We identified Ser¹², Thr²¹ (located in the BRCT domain) and Ser³⁷² (located in loop1) as the target residues. Mutation of these residues to alanine indicated that Ser³⁷² is the main phosphorylation site. Mobilization of loop1, which mediates DNA end micro-synapsis, is crucial both for terminal transferase and NHEJ. Interestingly, the phospho-mimicking S372E mutation specifically impaired these activities. Our evidences suggest that Polμ could be regulated in vivo by phosphorylation of the BRCT domain (Ser¹²/Thr²¹) and of Ser³⁷², affecting the function of loop1. Consequently, Polμ’s most distinctive activities would be turned off at specific cell-cycle phases (S and G2), when these promiscuous functions might be harmful to the cell.     

RNF8/RNF168信号通路在DNA损伤修复中的作用

细胞内DNA会受部分外界因素(如紫外辐射,电离辐射和化学毒素)和内部因素(如复制错误)的影响而发生损伤,包括DNA双链断裂、DNA错配和DNA交链等。DNA损伤发生后,损伤部位会被一些蛋白识别,进而招募一系列蛋白至损伤部位,形成一个修复系统。DNA双链断裂是最严重的一种DNA损伤,错误修复往往导致疾病的发生。DNA双链断裂(double strand break, DSB)后,细胞启动RNF8/RNF168信号通路进行修复。RNF8和RNF168是这条通路的枢纽蛋白;53BP和BRCA1是关键的效应蛋白,决定着DSB修复的方式;组蛋白泛素化、磷酸化和甲基化等翻译后修饰是这条通路顺利进行的基本条件;染色质重塑、泛素化酶/去泛素化酶平衡和蛋白稳定性是这条通路的主要调节方式。本综述对RNF8/RNF168信号通路进行了梳理总结,希望其能对相关研究者起到参考作用。     

Deubiquitinase USP2 stabilizes the MRE11–RAD50–NBS1 complex at DNA double-strand break sites by counteracting the ubiquitination of NBS1

The MRE11–RAD50–NBS1 (MRN) complex plays essential roles in the cellular response to DNA double-strand breaks (DSBs), which are the most cytotoxic DNA lesions, and is a target of various modifications and controls. Recently, lysine 48-linked ubiquitination of NBS1, resulting in premature disassembly of the MRN complex from DSB sites, was observed in cells lacking RECQL4 helicase activity. However, the role and control of this ubiquitination during the DSB response in cells with intact RECQL4 remain unknown. Here, we showed that USP2 counteracts this ubiquitination and stabilizes the MRN complex during the DSB response. By screening deubiquitinases that increase the stability of the MRN complex in RECQL4-deficient cells, USP2 was identified as a new deubiquitinase that acts at DSB sites to counteract NBS1 ubiquitination. We determined that USP2 is recruited to DSB sites in a manner dependent on ATM, a major checkpoint kinase against DSBs, and stably interacts with NBS1 and RECQL4 in immunoprecipitation experiments. Phosphorylation of two critical residues in the N terminus of USP2 by ATM is required for its recruitment to DSBs and its interaction with RECQL4. While inactivation of USP2 alone does not substantially influence the DSB response, we found that inactivation of USP2 and USP28, another deubiquitinase influencing NBS1 ubiquitination, results in premature disassembly of the MRN complex from DSB sites as well as defects in ATM activation and homologous recombination repair abilities. These results suggest that deubiquitinases counteracting NBS1 ubiquitination are essential for the stable maintenance of the MRN complex and proper cellular response to DSBs.     

Distinct roles for DNA-PK,ATM and ATR in RPA phosphorylation and checkpoint activation in response to replication stress

DNA damage encountered by DNA replication forks poses risks of genome destabilization, a precursor to carcinogenesis. Damage checkpoint systems cause cell cycle arrest, promote repair and induce programed cell death when damage is severe. Checkpoints are critical parts of the DNA damage response network that act to suppress cancer. DNA damage and perturbation of replication machinery causes replication stress, characterized by accumulation of single-stranded DNA bound by replication protein A (RPA), which triggers activation of ataxia telangiectasia and Rad3 related (ATR) and phosphorylation of the RPA32, subunit of RPA, leading to Chk1 activation and arrest. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) [a kinase related to ataxia telangiectasia mutated (ATM) and ATR] has well characterized roles in DNA double-strand break repair, but poorly understood roles in replication stress-induced RPA phosphorylation. We show that DNA-PKcs mutant cells fail to arrest replication following stress, and mutations in RPA32 phosphorylation sites targeted by DNA-PKcs increase the proportion of cells in mitosis, impair ATR signaling to Chk1 and confer a G2/M arrest defect. Inhibition of ATR and DNA-PK (but not ATM), mimic the defects observed in cells expressing mutant RPA32. Cells expressing mutant RPA32 or DNA-PKcs show sustained H2AX phosphorylation in response to replication stress that persists in cells entering mitosis, indicating inappropriate mitotic entry with unrepaired damage.     

The ionizing radiation-induced replication protein A phosphorylation response differs between ataxia telangiectasia and normal human cells.

Replication protein A (RPA), the trimeric single-stranded DNA-binding protein complex of eukaryotic cells, is important to DNA replication and repair. Phosphorylation of the p34 subunit of RPA is modulated by the cell cycle, occurring during S and G2 but not during G1. The function of phosphorylated p34 remains unknown. We show that RPA p34 phosphorylation is significantly induced by ionizing radiation. The phosphorylated form, p36, is similar if not identical to the phosphorylated S/G2 form. gamma-Irradiation-induced phosphorylation occurs without new protein synthesis and in cells in G1. Mutation of cdc2-type protein kinase phosphorylation sites in p34 eliminates the ionizing radiation response. The gamma-irradiation-induced phosphorylation of RPA p34 is delayed in cells from ataxia telangiectasia, a human inherited disease conferring DNA repair defects and early-onset tumorigenesis. UV-induced phosphorylation of RPA p34 occurs less rapidly than gamma-irradiation-induced phosphorylation but is kinetically similar between ataxia telangiectasia and normal cells. This is the first time that modification of a repair protein, RPA, has been linked with a DNA damage response and suggests that phosphorylation may play a role in regulating DNA repair pathways.     

A small ubiquitin binding domain inhibits ubiquitin-dependent protein recruitment to DNA repair foci

The rapid ubiquitination of chromatin surrounding DNA double-stranded breaks (DSB) drives the formation of large structures called ionizing radiation-induced foci (IRIF), comprising many DNA damage response (DDR) proteins. This process is regulated by RNF8 and RNF168 ubiquitin ligases and is thought to be necessary for DNA repair and activation of signaling pathways involved in regulating cell cycle checkpoints. Here we demonstrate that it is possible to interfere with ubiquitin-dependent recruitment of DDR factors by expressing proteins containing ubiquitin binding domains (UBDs) that bind to lysine 63-linked polyubiquitin chains. Expression of the E3 ubiquitin ligase RAD18 prevented chromatin spreading of 53BP1 at DSBs, and this phenomenon was dependent upon the integrity of the RAD18 UBD. An isolated RAD18 UBD interfered with 53BP1 chromatin spreading, as well as other important DDR mediators, including RAP80 and the BRCA1 tumor suppressor protein, consistent with the model that the RAD18 UBD is blocking access of proteins to ubiquitinated chromatin. Using the RAD18 UBD as a tool to impede localization of 53BP1 and BRCA1 to repair foci, we found that DDR signaling, DNA DSB repair, and radiosensitivity were unaffected. We did find that activated ATM (S1981P) and phosphorylated SMC1 (a specific target of ATM) were not detectable in DNA repair foci, in addition to upregulated homologous recombination repair, revealing 2 DDR responses that are dependent upon chromatin spreading of certain DDR factors at DSBs. These data demonstrate that select UBDs containing targeting motifs may be useful probes in determining the biological significance of protein–ubiquitin interactions.     

DYRK1A regulates the recruitment of 53BP1 to the sites of DNA damage in part through interaction with RNF169

Human Dual-specificity tyrosine (Y) Regulated Kinase 1A (DYRK1A) is encoded by a dosage dependent gene whereby either trisomy or haploinsufficiency result in developmental abnormalities. However, the function and regulation of this important protein kinase are not fully understood. Here, we report proteomic analysis of DYRK1A in human cells that revealed a novel role of DYRK1A in DNA double-strand breaks (DSBs) repair, mediated in part by its interaction with the ubiquitin-binding protein RNF169 that accumulates at the DSB sites and promotes homologous recombination repair (HRR) by displacing 53BP1, a key mediator of non-homologous end joining (NHEJ). We found that overexpression of active, but not the kinase inactive DYRK1A in U-2 OS cells inhibits accumulation of 53BP1 at the DSB sites in the RNF169-dependent manner. DYRK1A phosphorylates RNF169 at two sites that influence its ability to displace 53BP1 from the DSBs. Although DYRK1A is not required for the recruitment of RNF169 to the DSB sites and 53BP1 displacement, inhibition of DYRK1A or mutation of the DYRK1A phosphorylation sites in RNF169 decreases its ability to block accumulation of 53BP1 at the DSB sites. Interestingly, CRISPR-Cas9 knockout of DYRK1A in human and mouse cells also diminished the 53BP1 DSB recruitment in a manner that did not require RNF169, suggesting that dosage of DYRK1A can influence the DNA repair processes through both RNF169-dependent and independent mechanisms. Human U-2 OS cells devoid of DYRK1A display an increased HRR efficiency and resistance to DNA damage, therefore our findings implicate DYRK1A in the DNA repair processes.