MCL-1 localizes to sites of DNA damage and regulates DNA damage response

MCL-1, a pro-survival member of the BCL-2 family, was previously shown to have functions in ATR-dependent Chk1 phosphorylation following DNA damage. To further delineate these functions, we explored possible differences in DNA damage response caused by lack of MCL-1 in mouse embryo fibroblasts (MEFs). As expected, Mcl-1^-/- MEFs had delayed Chk1 phosphorylation following etoposide treatment, compared to wild type MEFs. However, their response to hydroxyurea, which causes a G1/S checkpoint response, was not significantly different. In addition, appearance of g-H2AX was delayed in the Mcl-1^-/- MEFs treated with etoposide. We next investigated whether MCL-1 is present, together with other DNA damage response proteins, at the sites of DNA damage. Immunoprecipitation of etoposide-treated extracts with anti-MCL-1 antibody showed association of MCL-1 with g-H2AX as well as NBS1. Immunofluorescent staining for MCL-1 further showed increased co-staining of MCL-1 and NBS1 following DNA damage. By using a system that creates DNA double strand breaks at specific sites in the genome, we demonstrated that MCL-1 is recruited directly adjacent to the sites of damage. Finally, in a direct demonstration of the importance of MCL-1 in allowing proper repair of DNA damage, we found that treatment for two brief exposures to etoposide over several days, which mimics the clinical situation of etoposide use, resulted in many more chromosomal abnormalities in the MEFs that lacked MCL-1. Together, these data indicate an important role for MCL-1 in coordinating DNA damage mediated checkpoint response, and have broad implications for the importance of MCL-1 in maintenance of genome integrity.     

MCL-1 localizes to sites of DNA damage and regulates DNA damage response

MCL-1, a pro-survival member of the BCL-2 family, was previously shown to have functions in ATR-dependent Chk1 phosphorylation following DNA damage. To further delineate these functions, we explored possible differences in DNA damage response caused by lack of MCL-1 in mouse embryo fibroblasts (MEFs). As expected, Mcl-1^-/- MEFs had delayed Chk1 phosphorylation following etoposide treatment, compared to wild type MEFs. However, their response to hydroxyurea, which causes a G₁/S checkpoint response, was not significantly different. In addition, appearance of γ-H2AX was delayed in the Mcl-1^-/- MEFs treated with etoposide. We next investigated whether MCL-1 is present, together with other DNA damage response proteins, at the sites of DNA damage. Immunoprecipitation of etoposide-treated extracts with anti-MCL-1 antibody showed association of MCL-1 with γ-H2AX as well as NBS1. Immunofluorescent staining for MCL-1 further showed increased co-staining of MCL-1 and NBS1 following DNA damage. By using a system that creates DNA double strand breaks at specific sites in the genome, we demonstrated that MCL-1 is recruited directly adjacent to the sites of damage. Finally, in a direct demonstration of the importance of MCL-1 in allowing proper repair of DNA damage, we found that treatment for two brief exposures to etoposide, followed by periods of recovery, which mimics the clinical situation of etoposide use, resulted in greater accumulation of chromosomal abnormalities in the MEFs that lacked MCL-1. Together, these data indicate an important role for MCL-1 in coordinating DNA damage mediated checkpoint response, and have broad implications for the importance of MCL-1 in maintenance of genome integrity.Key words: protein complex, DNA repair, checkpoint, G₂/M, chromosomes     

Proliferating cell nuclear antigen destabilizes c-Abl tyrosine kinase and regulates cell apoptosis in response to DNA damage

The tyrosine kinase, c-Abl, plays important roles in many aspects of cellular function. The activity of c-Abl is tightly controlled, but the underlying mechanism is unclear. Recent studies suggest that c-Abl function is regulated by distinct lipids in different cell types. In the present study, we show that the DNA replication factor, proliferating cell nuclear antigen (PCNA), interacts with c-Abl and destabilizes c-Abl by promoting its polyubiquitination and degradation. Moreover, deletion of a domain in c-Abl, the PIP box, disrupts its interaction with PCNA, abolishes the PCNA-induced degradation of nuclear c-Abl, and substantially increases the nuclear c-Abl apoptotic function. These findings indicate that PCNA negatively regulates the stability of c-Abl and thereby inhibits apoptosis in the response to DNA damage. Xiang He, Congwen Wei, and Ting Song contribute equally to this work. Wei Shi and Hui Zhong are co-correspondence authors.     

MDC1 regulates DNA-PK autophosphorylation in response to DNA damage

DNA damage initiates signaling events through kinase cascades that result in cell cycle checkpoint control and DNA repair. However, it is not yet clear how the signaling pathways relay to DNA damage repair. Using the repeat region of checkpoint protein MDC1 (mediator of DNA damage checkpoint protein 1), we identified DNA-PKcs/Ku as MDC1-associated proteins. Here, we show that MDC1 directly interacts with the Ku/DNA-PKcs complex. Down-regulation of MDC1 resulted in defective phospho-DNA-PKcs foci formation and DNA-PKcs autophosphorylation, suggesting that MDC1 regulates autophosphorylation of DNA-PKcs following DNA damage. Furthermore, DNA-PK-dependent DNA damage repair is defective in cells depleted of MDC1. Taken together, these results suggest that the MDC1 repeat region is involved in protein-protein interaction with DNA-PKcs/Ku, and MDC1 regulates DNA damage repair by influencing DNA-PK autophosphorylation. Therefore, MDC1 acts not only as a mediator of DNA damage checkpoint but also as a mediator of DNA damage repair.     

Ubiquitination regulates PTEN nuclear import and tumor suppression

The PTEN tumor suppressor is frequently affected in cancer cells, and inherited PTEN mutation causes cancer-susceptibility conditions such as Cowden syndrome. PTEN acts as a plasma-membrane lipid-phosphatase antagonizing the phosphoinositide 3-kinase/AKT cell survival pathway. However, PTEN is also found in cell nuclei, but mechanism, function, and relevance of nuclear localization remain unclear. We show that nuclear PTEN is essential for tumor suppression and that PTEN nuclear import is mediated by its monoubiquitination. A lysine mutant of PTEN, K289E associated with Cowden syndrome, retains catalytic activity but fails to accumulate in nuclei of patient tissue due to an import defect. We identify this and another lysine residue as major monoubiquitination sites essential for PTEN import. While nuclear PTEN is stable, polyubiquitination leads to its degradation in the cytoplasm. Thus, we identify cancer-associated mutations of PTEN that target its posttranslational modification and demonstrate how a discrete molecular mechanism dictates tumor progression by differentiating between degradation and protection of PTEN.     

Maf1 regulates intracellular lipid homeostasis in response to DNA damage response activation

Surveillance of DNA damage and maintenance of lipid metabolism are critical factors for general cellular homeostasis. We discovered that in response to DNA damage–inducing UV light exposure, intact Caenorhabditis elegans accumulate intracellular lipids in a dose-dependent manner. The increase in intracellular lipids in response to exposure to UV light utilizes mafr-1, a negative regulator of RNA polymerase III and the apical kinases atm-1 and atl-1 of the DNA damage response (DDR) pathway. In the absence of exposure to UV light, the genetic ablation of mafr-1 results in the activation of the DDR, including increased intracellular lipid accumulation, phosphorylation of ATM/ATR target proteins, and expression of the Bcl-2 homology region genes, egl-1 and ced-13. Taken together, our results reveal mafr-1 as a component the DDR pathway response to regulating lipid homeostasis following exposure to UV genotoxic stress.     

Involvement of nuclear domains in the cellular response to DNA damage

All cells of human organism are continuously damaged, and a damage of the genetic material can be especially dangerous. The reaction of the cell to DNA damage is a complex process, which includes damage signaling, repair, apoptosis or cell death. It is connected with serious changes in the cell nucleus, which are caused by posttranslational modifications and dynamic relocalizations of proteins as well as alterations in the expression of many genes. These changes are not limited to the sites of DNA damage, but involve whole cell nucleus, including its domains: PML bodies, nucleolus and Cajal bodies.     

Nup155 regulates nuclear envelope and nuclear pore complex formation in nematodes and vertebrates

Nuclear envelope (NE) formation during cell division in multicellular organisms is a central yet poorly understood biological process. We report that the conserved nucleoporin Nup155 has an essential function in NE formation in Caenorhabditis elegans embryos and in Xenopus laevis egg extracts. In vivo depletion of Nup155 led to failure of nuclear lamina formation and defects in chromosome segregation at anaphase. Nup155 depletion inhibited accumulation of nucleoporins at the nuclear periphery, including those recruited to chromatin early in NE formation. Electron microscopy analysis revealed that Nup155 is also required for the formation of a continuous nuclear membrane in vivo and in vitro. Time-course experiments indicated that Nup155 is recruited to chromatin at the time of NE sealing, suggesting that nuclear pore complex assembly has to progress to a relatively late stage before NE membrane assembly occurs.     

NEDDylation regulates RAD18 ubiquitination and localization in response to oxidative DNA damage

Genome integrity is important for cell growth, development and proliferation. The E3 ligase RAD18 plays a vital role in the DNA damage response (DDR) to maintain genome integrity. Recent studies reveal that RAD18 has non-ubiquitinated and mono-ubiquitinated form in normal cells. However, whether RAD18 undergoes other post-translational modification remains to be investigated. Here we show that RAD18 is a target of NEDD8, an ubiquitin-like protein. In response to hydrogen peroxide (H₂O₂)-induced oxidative stress, RAD18 NEDDylation increases significantly, while its ubiquitination decreases. Moreover, NEDD8 overexpression or deNEDDylase NEDP1 deletion further antagonizes RAD18 ubiquitination. In addition, treatment with MLN4924, an inhibitor of NEDD8-activating Enzyme, reduces the interaction between PCNA and RAD18, which blocks the localization of RAD18 to form foci, and thus inhibiting polymerase η recruitment after oxidative stress. Together, our study demonstrates that RAD18 NEDDylation regulates its localization and involves in the DDR pathway by modulating RAD18 ubiquitination.     

A novel ATM-dependent pathway regulates protein phosphatase 1 in response to DNA damage

Protein phosphatase 1 (PP1), a major protein phosphatase important for a variety of cellular responses, is activated in response to ionizing irradiation (IR)-induced DNA damage. Here, we report that IR induces the rapid dissociation of PP1 from its regulatory subunit inhibitor-2 (I-2) and that the process requires ataxia-telangiectasia mutated (ATM), a protein kinase central to DNA damage responses. In response to IR, ATM phosphorylates I-2 on serine 43, leading to the dissociation of the PP1-I-2 complex and the activation of PP1. Furthermore, ATM-mediated I-2 phosphorylation results in the inhibition of the Aurora-B kinase, the down-regulation of histone H3 serine 10 phosphorylation, and the activation of the G(2)/M checkpoint. Collectively, the results of these studies demonstrate a novel pathway that links ATM, PP1, and I-2 in the cellular response to DNA damage.     

JNK phosphorylation of 14-3-3 proteins regulates nuclear targeting of c-Abl in the apoptotic response to DNA damage

The ubiquitously expressed c-Abl tyrosine kinase localizes to the cytoplasm and nucleus. Nuclear c-Abl is activated by diverse genotoxic agents and induces apoptosis; however, the mechanisms that are responsible for nuclear targeting of c-Abl remain unclear. Here, we show that cytoplasmic c-Abl is targeted to the nucleus in the DNA damage response. The results show that c-Abl is sequestered into the cytoplasm by binding to 14-3-3 proteins. Phosphorylation of c-Abl on Thr 735 functions as a site for direct binding to 14-3-3 proteins. We also show that, in response to DNA damage, activation of the c-Jun N-terminal kinase (Jnk) induces phosphorylation of 14-3-3 proteins and their release from c-Abl. Together with these results, expression of an unphosphorylated 14-3-3 mutant attenuates DNA-damage-induced nuclear import of c-Abl and apoptosis. These findings indicate that 14-3-3 proteins are pivotal regulators of intracellular c-Abl localization and of the apoptotic response to genotoxic stress.     

PARP activation regulates the RNA-binding protein NONO in the DNA damage response to DNA double-strand breaks

After the generation of DNA double-strand breaks (DSBs), poly(ADP-ribose) polymerase-1 (PARP-1) is one of the first proteins to be recruited and activated through its binding to the free DNA ends. Upon activation, PARP-1 uses NAD⁺ to generate large amounts of poly(ADP-ribose) (PAR), which facilitates the recruitment of DNA repair factors. Here, we identify the RNA-binding protein NONO, a partner protein of SFPQ, as a novel PAR-binding protein. The protein motif being primarily responsible for PAR-binding is the RNA recognition motif 1 (RRM1), which is also crucial for RNA-binding, highlighting a competition between RNA and PAR as they share the same binding site. Strikingly, the in vivo recruitment of NONO to DNA damage sites completely depends on PAR, generated by activated PARP-1. Furthermore, we show that upon PAR-dependent recruitment, NONO stimulates nonhomologous end joining (NHEJ) and represses homologous recombination (HR) in vivo. Our results therefore place NONO after PARP activation in the context of DNA DSB repair pathway decision. Understanding the mechanism of action of proteins that act in the same pathway as PARP-1 is crucial to shed more light onto the effect of interference on PAR-mediated pathways with PARP inhibitors, which have already reached phase III clinical trials but are until date poorly understood.     

Tyrosine 370 phosphorylation of ATM positively regulates DNA damage response

Ataxia telangiectasia mutated (ATM) mediates DNA damage response by controling irradiation-induced foci formation, cell cycle checkpoint, and apoptosis. However, how upstream signaling regulates ATM is not completely understood. Here, we show that upon irradiation stimulation, ATM associates with and is phosphorylated by epidermal growth factor receptor (EGFR) at Tyr370 (Y370) at the site of DNA double-strand breaks. Depletion of endogenous EGFR impairs ATM-mediated foci formation, homologous recombination, and DNA repair. Moreover, pretreatment with an EGFR kinase inhibitor, gefitinib, blocks EGFR and ATM association, hinders CHK2 activation and subsequent foci formation, and increases radiosensitivity. Thus, we reveal a critical mechanism by which EGFR directly regulates ATM activation in DNA damage response, and our results suggest that the status of ATM Y370 phosphorylation has the potential to serve as a biomarker to stratify patients for either radiotherapy alone or in combination with EGFR inhibition.     

SOCS3 regulates p21 expression and cell cycle arrest in response to DNA damage

Genotoxic agents such as ionizing radiation trigger cell cycle arrest at the G1/S and G2/M checkpoints, allowing cells to repair damaged DNA before entry into mitosis. DNA damage-induced G1 arrest involves p53-dependent expression of p21 (Cip1/Waf-1), which inhibits cyclin-dependent kinases and blocks S phase entry. While much of the core DNA damage response has been well-studied, other signaling proteins that intersect with and modulate this response remain uncharacterized. In this study, we identify Suppressor of Cytokine Signaling (SOCS)-3 as an important regulator of radiation-induced G1 arrest. SOCS3-deficient fibroblasts fail to undergo G1 arrest and accumulate in the G2/M phase of the cell cycle. SOCS3 knockout cells phosphorylate p53 and H2AX normally in response to radiation, but fail to upregulate p21 expression. In addition, STAT3 phosphorylation is elevated in SOCS3-deficient cells compared to WT cells. Normal G1 arrest can be restored in SOCS3 KO cells by retroviral transduction of WT SOCS3 or a dominant-negative mutant of STAT3. Our results suggest a novel function for SOCS3 in the control of genome stability by negatively regulating STAT3-dependent radioresistant DNA synthesis, and promoting p53-dependent p21 expression.     

NEDDylation antagonizes ubiquitination of proliferating cell nuclear antigen and regulates the recruitment of polymerase η in response to oxidative DNA damage

NEDDylation has been shown to participate in the DNA damage pathway, but the substrates of neural precursor cell expressed developmentally downregulated 8 (NEDD8) and the roles of NEDDylation involved in the DNA damage response (DDR) are largely unknown. Translesion synthesis (TLS) is a damage-tolerance mechanism, in which RAD18/RAD6-mediated monoubiquitinated proliferating cell nuclear antigen (PCNA) promotes recruitment of polymerase η (polη) to bypass lesions. Here we identify PCNA as a substrate of NEDD8, and show that E3 ligase RAD18-catalyzed PCNA NEDDylation antagonizes its ubiquitination. In addition, NEDP1 acts as the deNEDDylase of PCNA, and NEDP1 deletion enhances PCNA NEDDylation but reduces its ubiquitination. In response to H₂O₂ stimulation, NEDP1 disassociates from PCNA and RAD18-dependent PCNA NEDDylation increases markedly after its ubiquitination. Impairment of NEDDylation by Ubc12 knockout enhances PCNA ubiquitination and promotes PCNA-polη interaction, while up-regulation of NEDDylation by NEDD8 overexpression or NEDP1 deletion reduces the excessive accumulation of ubiquitinated PCNA, thus inhibits PCNA-polη interaction and blocks polη foci formation. Moreover, Ubc12 knockout decreases cell sensitivity to H₂O₂-induced oxidative stress, but NEDP1 deletion aggravates this sensitivity. Collectively, our study elucidates the important role of NEDDylation in the DDR as a modulator of PCNA monoubiquitination and polη recruitment.     

AtMMS21 regulates DNA damage response and homologous recombination repair in Arabidopsis

DNA damage is a significant problem in living organisms and DNA repair pathways have been evolved in different species to maintain genomic stability. Here we demonstrated the molecular function of AtMMS21, a component of SMC5/6 complex, in plant DNA damage response. Compared with wild type, the AtMMS21 mutant plants show hypersensitivity in the DNA damaging treatments by MMS, cisplatin and gamma radiation. However, mms21-1 is not sensitive to replication blocking agents hydroxyurea and aphidicolin. The expression of a DNA damage response gene PARP2 is upregulated in mms21-1 under normal condition, suggesting that this signaling pathway is constitutively activated in the mutant. Depletion of ATAXIA-TELANGIECTASIA MUTATED (ATM) in mms21-1 enhances its root growth defect phenotype, indicating that ATM and AtMMS21 may play additive roles in DNA damage pathway. The analysis of homologous recombination frequency showed that the number of recombination events is reduced in mms21-1 mutant. Conclusively, we provided evidence that AtMMS21 plays an important role in homologous recombination for DNA damage repair.     

USP37 regulates DNA damage response through stabilizing and deubiquitinating BLM

The human RecQ helicase BLM is involved in the DNA damage response, DNA metabolism, and genetic stability. Loss of function mutations in BLM cause the genetic instability/cancer predisposition syndrome Bloom syndrome. However, the molecular mechanism underlying the regulation of BLM in cancers remains largely elusive. Here, we demonstrate that the deubiquitinating enzyme USP37 interacts with BLM and that USP37 deubiquitinates and stabilizes BLM, thereby sustaining the DNA damage response (DDR). Mechanistically, DNA double-strand breaks (DSB) promotes ATM phosphorylation of USP37 and enhances the binding between USP37 and BLM. Moreover, knockdown of USP37 increases BLM polyubiquitination, accelerates its proteolysis, and impairs its function in DNA damage response. This leads to enhanced DNA damage and sensitizes breast cancer cells to DNA-damaging agents in both cell culture and in vivo mouse models. Collectively, our results establish a novel molecular mechanism for the USP37–BLM axis in regulating DSB repair with an important role in chemotherapy and radiotherapy response in human cancers.