Acquisition of meiotic DNA repair regulators maintain genome stability in glioblastoma

Glioblastoma (GBM), the most prevalent type of primary intrinsic brain cancer in adults, remains universally fatal despite maximal therapy, including radiotherapy and chemotherapy. Cytotoxic therapy generates double-stranded DNA breaks (DSBs), most commonly repaired by homologous recombination (HR). We hypothesized that cancer cells coopt meiotic repair machinery as DSBs are generated during meiosis and repaired by molecular complexes distinct from genotoxic responses in somatic tissues. Indeed, we found that gliomas express meiotic repair genes and their expression informed poor prognosis. We interrogated the function of disrupted meiotic cDNA1 (DMC1), a homolog of RAD51, the primary recombinase used in mitotic cells to search and recombine with the homologous DNA template. DMC1, whose only known function is as an HR recombinase, was expressed by GBM cells and induced by radiation. Although targeting DMC1 in non-neoplastic cells minimally altered cell growth, DMC1 depletion in GBM cells decreased proliferation, induced activation of CHK1 and expression of p21^CIP1/WAF1, and increased RPA foci, suggesting increased replication stress. Combining loss of DMC1 with ionizing radiation inhibited activation of DNA damage responses and increased radiosensitivity. Furthermore, loss of DMC1 reduced tumor growth and prolonged survival in vivo. Our results suggest that cancers coopt meiotic genes to augment survival under genotoxic stress, offering molecular targets with high therapeutic indices.Glioblastomas (GBMs) rank among the deadliest of all human cancers, with only modest improvement in patient survival over recent decades. More than 12 000 GBM patients are diagnosed annually in the United States.^{1, 2} Despite aggressive treatment consisting of maximal safe surgical resection, concurrent radiotherapy and chemotherapy, and adjuvant chemotherapy, median survival remains dismal at 12–15 months.^{3, 4} Although numerous molecular targets have been identified in GBM, no molecularly targeted therapy has demonstrated a survival benefit. Radiotherapy remains the cornerstone of post-surgical GBM therapy with modest additional benefit offered by concurrent administration of the oral methylator, temozolomide. However, radioresistance and tumor recurrence is universal in GBM.^{4, 5, 6} Radiation also damages non-neoplastic brain tissue, resulting in cognitive impairment and decreased quality-of-life.⁷ Focal high-dose radiation reduces toxicity to non-neoplastic tissue, but tumor invasion into normal brain regions limits the survival benefit of highly focused radiotherapy techniques, like gamma knife and proton beam, establishing a need for improved combinatorial treatments, such as radiosensitizers.^{8, 9} To date, no radiosensitizer has successfully increased survival with acceptable toxicity in a clinical trial. Based on this background, we sought novel molecular targets that mediate responses to genotoxic stress and have limited function in normal cells.During mitosis, cells inspect the integrity of their DNA and repair replication errors through cell-state and error-specific mechanisms.¹⁰ Unrepaired or large regions of DNA damage overwhelm replication mechanisms to induce cell death.^{10, 11} DNA double-strand breaks (DSBs) are detrimental as they cause large-scale chromosomal rearrangements.¹⁰ The homologous recombination (HR) pathway is primarily used to repair DSBs during S- and G₂-phases, providing access to both sister and homologous chromosomes as repair templates.^{7, 12} RADiation sensitive 51 (RAD51) is a key recombinase important in HR and replication fork maintenance, functioning in both mitotic and meiotic cells.^{7, 12, 13, 14, 15} Phosphorylated RAD51 replaces replication protein A (RPA) upon DNA loading.¹⁶ Recombination mediated by RAD51 with the intact DNA template strand results in a relatively error-free repair.¹²In contrast to mitosis, germ cells undergoing meiosis actively generate genetic diversity through induction of programmed DSBs, which are repaired through HR.^{17, 18, 19} In meiotic HR, RAD51 functions in conjunction with the meiosis-specific recombinase, disrupted meiotic cDNA1 (DMC1). RAD51 and DMC1 are loaded onto DNA by a meiosis-specific accessory protein complex, homologous-pairing protein 2 (HOP2)–meiotic nuclear divisions 1 (MND1), to promote homologous strand invasion and dissociation-loop (D-loop) formation.^{20, 21} D-loops formed using the DMC1–RAD51 complex are more resistant to dissociation as opposed to D-loops formed by RAD51 alone, increasing the likelihood of DNA crossover events.²⁰ In addition, DMC1-directed crossovers preferentially utilize the homologous chromosome further increasing genetic variation.²²GBM cells commonly harbor genetic lesions that promote unrestrained proliferation but also stimulate genotoxic stress responses. Neoplastic cells do not require perfect fidelity of repair. In fact, dysfunctional repair accelerates genetic evolution of clones, but cancer cells must acquire mechanisms to bypass cell death or senescence in response to exogenous stressors.^{11, 23} Radiotherapy targets proliferating cancer cells by production of reactive oxygen species, leading to generation of DSBs and activation of the DNA damage response (DDR) pathway.^{11, 24} DSBs generated as a result of ionizing radiation (IR) are repaired through HR or non-homologous end joining (NHEJ).^{7, 12, 25, 26} Terminally differentiated neurons are post-mitotic and rely on NHEJ as a means to repair DNA DSBs. Therefore, inhibition of the NHEJ pathway may result in unfavorable normal neural cell toxicity.²⁶The HR pathway is an attractive target as it is linked to increased genetic variation and loss of heterozygosity (LOH).^{12, 27} Multiple HR checkpoints have been proposed as potential therapeutic targets for GBM.^{28, 29, 30, 31} Although the prognostic value of RAD51 expression in GBM is unresolved,^{29, 32, 33} RAD51 is consistently elevated in GBM compared with normal brain.³³ Reducing RAD51 expression radiosensitizes GBM cells,²⁹ but may have a limited therapeutic index because of the potentially toxic effects on non-neoplastic cells. In this study, we investigated the aberrant activity of meiotic HR regulators in glioma, focusing on the meiosis-specific DMC1. Activation of meiotic repair genes in neoplastic cells selectively provides tumor cells with a repair mechanism to evade cell death caused by DNA damage, yet increase genetic diversity to drive clonal evolution.     

Platelet-activating factor induces cell cycle arrest and disrupts the DNA damage response in mast cells

Platelet-activating factor (PAF) is a potent phospholipid modulator of inflammation that has diverse physiological and pathological functions. Previously, we demonstrated that PAF has an essential role in ultraviolet (UV)-induced immunosuppression and reduces the repair of damaged DNA, suggesting that UV-induced PAF is contributing to skin cancer initiation by inducing immune suppression and also affecting a proper DNA damage response. The exact role of PAF in modulating cell proliferation, differentiation or transformation is unclear. Here, we investigated the mechanism(s) by which PAF affects the cell cycle and impairs early DNA damage response. PAF arrests proliferation in transformed and nontransformed human mast cells by reducing the expression of cyclin-B1 and promoting the expression of p21. PAF-treated cells show a dose-dependent cell cycle arrest mainly at G2–M, and a decrease in the DNA damage response elements MCPH1/BRIT-1 and ataxia telangiectasia and rad related (ATR). In addition, PAF disrupts the localization of p-ataxia telangiectasia mutated (p-ATM), and phosphorylated-ataxia telangiectasia and rad related (p-ATR) at the site of DNA damage. Whereas the potent effect on cell cycle arrest may imply a tumor suppressor activity for PAF, the impairment of proper DNA damage response might implicate PAF as a tumor promoter. The outcome of these diverse effects may be dependent on specific cues in the microenvironment.Ultraviolet (UV)-mediated immunosuppression poses a major risk for skin cancer induction,^{1, 2} and many have reported that an essential mediator in this process is UV-induced platelet-activating factor (PAF; 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine).^{3, 4, 5} PAF is a phospholipid, first discovered as a secreted component by activated innate immune cells,^{6, 7} that mediates its activity by binding to a G-protein-coupled receptor.⁸ It is involved in a variety of mechanisms including the release of histamine in activated leukocytes,^{9, 10, 11} anaphylaxis, and phagocytosis.¹²Exposure to low doses of UV radiation activates PAF release by keratinocytes,^{13, 14} so it is likely that most of the population is regularly exposed to keratinocyte-derived PAF. In previous studies we showed that PAF upregulates both CXCR4 on mast cells and its ligand (CXCL12) on draining lymph node cells, promoting the migration of dermal mast cells from inflamed skin to the lymph nodes.¹⁵ Mast cells that reach the draining lymph nodes activate immune suppression by releasing interleukin 10.¹⁶ Blocking mast cell migration by using a CXCR4 antagonist, AMD3100, blocks UV-induced immune suppression and the induction of skin cancer.^{15, 17} No immune suppression is noted when PAF receptor-deficient mice (PAFR^-/-) are exposed to UV radiation,^{4, 5} nor can one reconstitute immune suppression when PAFR^-/- mast cells are used to reconstitute mast cell-deficient mice.¹⁸ PAF also has a critical role in skin cancer induction and progression,^{19, 20} and this may reflect its capacity to both induce immune suppression and hamper DNA repair.²¹Hanahan and Weinberg recognized the important roles inflammation and immune evasion play in the initiation of cancer.²² UV-induced PAF by activating immune suppression, retarding DNA repair and activating inflammation clearly constitutes an important hallmark for cancer induction. Supporting this idea is the observation that PAF is involved in a variety of other cancers besides skin cancer.^{23, 24, 25, 26, 27} Although we previously demonstrated that PAF suppresses the rate of DNA repair in vivo,²¹ little is known regarding the mechanisms involved. In this study we performed a series of experiments to determine how PAF affects DNA repair by examining important checkpoints that regulate DNA repair and cell cycle progression. We primarily used mast cells because of the critical role these cells have in UV-induced immune suppression and skin cancer induction,^{15, 28} and also because the dermis where they reside is targeted by UV-induced PAF.¹⁸     

Restoration of miR-101 suppresses lung tumorigenesis through inhibition of DNMT3a-dependent DNA methylation

The deregulation of miR-101 and DNMT3a has been implicated in the pathogenesis of multiple tumor types, but whether and how miR-101 silencing and DNMT3a overexpression contribute to lung tumorigenesis remain elusive. Here we show that miR-101 downregulation associates with DNMT3a overexpression in lung cancer cell lines and patient tissues. Ectopic miR-101 expression remarkably abrogated the DNMT3a 3′-UTR luciferase activity corresponding to the miR-101 binding site and caused an attenuated expression of endogenous DNMT3a, which led to a reduction of global DNA methylation and the re-expression of tumor suppressor CDH1 via its promoter DNA hypomethylation. Functionally, restoration of miR-101 expression suppressed lung cancer cell clonability and migration, which recapitulated the DNMT3a knockdown effects. Interestingly, miR-101 synergized with decitabine to downregulate DNMT3a and to reduce DNA methylation. Importantly, ectopic miR-101 expression was sufficient to trigger in vivo lung tumor regression and the blockage of metastasis. Consistent with these phenotypes, examination of xenograft tumors disclosed an increase of miR-101, a decrease of DNMT3a and the subsequent DNA demethylation. These findings support that the loss or suppression of miR-101 function accelerates lung tumorigenesis through DNMT3a-dependent DNA methylation, and suggest that miR-101-DNMT3a axis may have therapeutic value in treating refractory lung cancer.Owing to a high propensity for recurrence and a high rate of metastasis at the advanced stages,^{1, 2, 3} lung cancer remains the leading cause of cancer-related mortality. DNA methylation is a major epigenetic rule controlling chromosomal stability and gene expression.^{4, 5} It is under control of DNA methyltransferases (DNMTs), whose overexpression in lung cancer cells predicts worse outcomes.^{6, 7} It is postulated that DNMT overexpression induces DNA hypermethylation and silencing of tumor suppressor genes (TSGs), leading to an aggressive lung cancer. Indeed, enforced expression of DNMT1 or DNMT3a increases DNA methylation, while the abolition of DNMT expression by genetic depletion, microRNAs (miRs) or small molecules reduces genome-wide and gene-specific DNA methylation and restores TSG expression.^{8, 9, 10, 11, 12, 13} As TSGs are the master controllers for cell multiplicity and their silencing predicts poor prognosis,^{14, 15} TSG re-expression via promoter DNA hypomethylation inhibits cell proliferation and induces cell differentiation.^{13, 16} Thus, DNMT gene abundance could serve as a target for anticancer therapy, but how DNMT upregulation occurs in lung cancer is incompletely understood.MiRs are small non-coding RNAs that crucially regulate target gene expression. Up to 30% of all protein-coding genes are predicted to be targeted by miRs,^{17, 18} supporting the key roles of miRs in controlling cell fate.^{19, 20, 21, 22} Research is showing that certain miRs are frequently dysregulated in cancers, including lung cancer.^{7, 23, 24} As miR targets can promote or inhibit cancer cell expansion, miRs have huge potential for acting as bona fide oncogenes (i.e., miR-21) or TSGs (i.e., miR-29b).^{7, 25} We and others demonstrated that the levels of DNMT1 or DNMT3a or DNMT3b are regulated by miR-29b, miR-148, miR-152 or miR-30c,^{7, 13, 26, 27} and overexpression of these miRs results in DNA hypomethylation and TSG reactivation with the concurrent blockage of cancer cell proliferation.^{7, 13} These findings underscore the importance of miRs as epigenetic modulators and highlight their therapeutic applications.MiR-101 is frequently silenced in human cancers^{28, 29, 30, 31} and, importantly, exhibits antitumorigenic properties when overexpressed. Mechanistically, miR-101 inactivation by genomic loss causes the overexpression of EZH2, a histone methyltransferase, via 3′-UTR targeting, which is followed by histone hypermethylation and aggressive tumorigenesis.^{29, 30, 32} However, whether and how miR-101 silencing contributes to DNA hypermethylation patterning in lung cancer is unclear. In this study, we explore the role of miR-101 in regulating DNMT3a expression and the impacts of miR-101-DNMT3a nexus on lung cancer pathogenesis. We showed that the expression of miR-101 and DNMT3a was negatively correlated in lung cancer. We presented evidence that ectopic miR-101 expression decreased DNMT3a levels, reduced global DNA methylation and upregulated CDH1 via its promoter DNA demethylation. The biological significance of miR-101-mediated DNA hypomethylation and CDH1 re-expression was evident by its inhibition of lung tumor cell growth in vitro and in vivo. Thus, our findings mechanistically and functionally link miR-101 silencing to DNA hypermethylation in lung cancer cells.     

JWA reverses cisplatin resistance via the CK2—XRCC1 pathway in human gastric cancer cells

Gastric cancer is the third most common malignancy in China, with a median 5-year survival of only 20%. Cisplatin has been used in first-line cancer treatment for several types of cancer including gastric cancer. However, patients are often primary resistant or develop acquired resistance resulting in relapse of the cancer and reduced survival. Recently, we demonstrated that the reduced expression of base excision repair protein XRCC1 and its upstream regulator JWA in gastric cancerous tissues correlated with a significant survival benefit of adjuvant first-line platinum-based chemotherapy as well as XRCC1 playing an important role in the DNA repair of cisplatin-resistant gastric cancer cells. In the present study, we demonstrated the role of JWA in cisplatin-induced DNA lesions and aquired cisplatin resistance in five cell-culture models: gastric epithelial cells GES-1, cisplatin-sensitive gastric cancer cell lines BGC823 and SGC7901, and the cisplatin-resistant gastric cancer cell lines BGC823/DDP and SGC7901/DDP. Our results indicated that JWA is required for DNA repair following cisplatin-induced double-strand breaks (DSBs) via XRCC1 in normal gastric epithelial cells. However, in gastric cancer cells, JWA enhanced cisplatin-induced cell death through regulation of DNA damage-induced apoptosis. The protein expression of JWA was significantly decreased in cisplatin-resistant cells and contributed to cisplatin resistance. Interestingly, as JWA upregulated XRCC1 expression in normal cells, JWA downregulated XRCC1 expression through promoting the degradation of XRCC1 in cisplatin-resistant gastric cancer cells. Furthermore, the negative regulation of JWA to XRCC1 was blocked due to the mutation of 518S/519T/523T residues of XRCC1, and indicating that the CK2 activated 518S/519T/523T phosphorylation is a key point in the regulation of JWA to XRCC1. In conclusion, we report for the first time that JWA regulated cisplatin-induced DNA damage and apoptosis through the CK2—P-XRCC1—XRCC1 pathway, indicating a putative drug target for reversing cisplatin resistance in gastric cancer.Gastric cancer (GC) is the fifth most common human malignant tumor worldwide but third cause of cancer death.¹ In 2012, there were 405 000 new GC cases diagnosed and 325 000 deaths in China.¹ Current strategy for treatment of GC includes surgery with chemotherapy for potentially curable disease and chemotherapy only for advanced disease. Unfortunately, owing to intrinsic or acquired drug resistance, relapse and metastasis are common and result in high mortality of GC.²Cisplatin is a widely used chemotherapeutic drug for treating various tumors including GC.³ Cisplatin triggers apoptosis by inducing DNA damage through crosslinking of the DNA.⁴ However, cancer cells often develop multiple mechanisms to overcome cisplatin-induced DNA damage and apoptosis, and lead to cisplatin resistance.^{5, 6} Two of the major systems activated are enhanced capability of DNA repair and anti-apoptosis signaling pathways.^{7, 8}XRCC1 is a key mediator of single-strand break DNA repair, and is involved in the process of cisplatin-induced DNA damage repair in various tumors.^{9, 10, 11} XRCC1 was found to identify and bind to DNA interstrand crosslinks induced by cisplatin.¹² Moreover casein kinase 2 (CK2) phosphorylates XRCC1 and is required for its stability and efficient DNA repair.¹³ A selective small molecule inhibitor of CK2, CX-4945, was found to block the cisplatin-induced DNA repair response by decreasing the phosphorylation of XRCC1 at CK2-specific phosphorylation sites.¹⁴ This body of evidence indicates a critical role of XRCC1 and CK2 in cisplatin resistance.The JWA gene, also known as ARL6ip5, was initially cloned from human tracheal bronchial epithelial cells after treatment with all-trans retinoic acid.¹⁵ Subsequent studies indicated that JWA is involved in the cellular responses to heat shock and chemical-mediated oxidative stresses.^{16, 17} Moreover, JWA functions as a base excision repair protein in oxidative-stress-induced DNA single-strand breaks in NIH-3T3 and HELF cells, as evidenced by the positive regulation of XRCC1 levels through MAPK signal pathway and protecting XRCC1 protein from ubiquitination and degradation by proteasome.^{18, 19} However, JWA is also a structurally novel microtubule-binding protein, which regulates cancer cell migration via MAPK cascades and mediates differentiation of leukemic cells.^{20, 21, 22} JWA significantly inhibits melanoma adhesion, invasion and metastasis via integrin aVb3 signaling.²³ More recent data have shown that JWA is required for As₂O₃-induced apoptosis in HeLa and MCF-7 cells via reactive oxygen species and mitochondria-linked signal pathway or promoted p38 MAPK-linked tubulin polymerization.^{24, 25} These reports indicate that the JWA functions as a tumor suppressor for tumor initiation and development.Recently, we reported the prognostic and predictive role of JWA and XRCC1 expression in GC. JWA and XRCC1 protein levels are significantly downregulated in GC lesions compared with adjacent noncancerous tissues, whereas platinum-based chemotherapy significantly improved overall survival in GC patients with low levels of tumoral JWA or XRCC1 expression.²⁶ Subsequent studies indicated that overexpression of XRCC1 contributed to cisplatin resistance in GC cells and showed that XRCC1 protein was important for effective repair of cisplatin-induced DSBs in GC cells.²⁷ However, the contribution of JWA to cisplatin resistance in GC and underlying mechanisms are not fully understood.The objectives of the present study were to investigate the role of JWA in cisplatin resistance of GC cells and elucidate the underlying mechanisms of action. Our results demonstrated that JWA negatively regulated XRCC1 through the CK2—p-XRCC1 pathway in cisplatin-resistant GC cells. The JWA could be a valuable target for reversal of cisplatin resistance in human GC.     

XLS (c9orf142) is a new component of mammalian DNA double-stranded break repair

Repair of double-stranded DNA breaks (DSBs) in mammalian cells primarily occurs by the non-homologous end-joining (NHEJ) pathway, which requires seven core proteins (Ku70/Ku86, DNA-PKcs (DNA-dependent protein kinase catalytic subunit), Artemis, XRCC4-like factor (XLF), XRCC4 and DNA ligase IV). Here we show using combined affinity purification and mass spectrometry that DNA-PKcs co-purifies with all known core NHEJ factors. Furthermore, we have identified a novel evolutionary conserved protein associated with DNA-PKcs—c9orf142. Computer-based modelling of c9orf142 predicted a structure very similar to XRCC4, hence we have named c9orf142—XLS (XRCC4-like small protein). Depletion of c9orf142/XLS in cells impaired DSB repair consistent with a defect in NHEJ. Furthermore, c9orf142/XLS interacted with other core NHEJ factors. These results demonstrate the existence of a new component of the NHEJ DNA repair pathway in mammalian cells.Double-stranded DNA breaks (DSBs) are among the most cytotoxic DNA lesions for mammalian cells.¹ Effective repair of DSBs is essential for cellular survival and for suppression of potential deleterious chromosomal rearrangements.² Two main DNA repair pathways eliminate DSBs—homologous recombination (HR) or non-homologous end joining (NHEJ). HR utilises an undamaged copy of the chromosome as a template to direct repair, thus this restricts HR to the S and G2/M phases of the cell cycle, when such an extra chromosome copy is available.³ NHEJ performs the bulk of DSB repair in mammalian cells and in particular in during the G1 phase of the cell cycle, where the cells are completely dependent on NHEJ. NHEJ can be further subdivided into so-called classical NHEJ (c-NHEJ) and alternative NHEJ (alt-NHEJ).⁴ These DNA repair pathways utilise distinct protein components and also show different efficiencies of end ligation. In general, c-NHEJ is much more effective in end ligation than alt-NHEJ and can ligate most unrelated DNA ends directly or with minimal processing. In contrast alt-NHEJ requires short microhomologies between the DNA ends for ligation.⁵ C-NHEJ requires the following seven core proteins: Ku70/Ku86 dimers, DNA-PKcs (DNA-dependent protein kinase catalytic subunit), Artemis nuclease, XRCC4-like factor (XLF) and the XRCC4/ligase IV complex.^{6, 7} The DSB repair during c-NHEJ is initiated by the Ku dimer that senses the presence of free double-stranded DNA ends in cells and rapidly binds such ends with high affinity. DNA-bound Ku then recruits DNA-PKcs (DNA-PKcs/Ku70/Ku86 complex is termed DNA-PK holoenzyme), which has a protein kinase activity and is required for activation of the nuclease Artemis.⁸ Artemis, in turn, is responsible for DNA end processing in order to achieve DNA end structures suitable for ligation. The final step of c-NHEJ is the ligation of processed DNA ends by XRCC4/ligase IV complex. This final step is stimulated by XLF protein that interacts with XRCC4 forming long filamentous structures at DSBs to facilitate DNA end joining.^{9, 10} XRCC4 and XLF factors are distinct among NHEJ factors in that they share similar tertiary structure but show low primary sequence conservation.¹¹ Since the identification of XLF in 2006, no new core factors have been discovered.^{11, 12} Importantly, c-NHEJ is essential for proper development, as mutations in this pathway lead to immunodeficiency and defective neurogenesis in humans.⁷ It is therefore essential to fully decipher the identity of components for the c-NHEJ pathway and their regulation.In this study, proteomic analysis of DNA-PKcs-containing protein complexes identified an abundant previously uncharacterised protein c9orf142, which we have named c9orf142—XLS (XRCC4-like small protein). Structural modelling predicts XLS to be highly similar to XRCC4 and XLF, and depletion of XLS delays ionising radiation (IR)-induced DNA DSB repair. Moreover, XLS is associated with other core c-NHEJ factors. Our data strongly suggest that c9orf142/XLS represents a novel c-NHEJ component in mammalian cells.     

The Exonuclease Homolog OsRAD1 Promotes Accurate Meiotic Double-Strand Break Repair by Suppressing Nonhomologous End Joining

During meiosis, programmed double-strand breaks (DSBs) are generated to initiate homologous recombination, which is crucial for faithful chromosome segregation. In yeast, Radiation sensitive1 (RAD1) acts together with Radiation sensitive9 (RAD9) and Hydroxyurea sensitive1 (HUS1) to facilitate meiotic recombination via cell-cycle checkpoint control. However, little is known about the meiotic functions of these proteins in higher eukaryotes. Here, we characterized a RAD1 homolog in rice (Oryza sativa) and obtained evidence that O. sativa RAD1 (OsRAD1) is important for meiotic DSB repair. Loss of OsRAD1 led to abnormal chromosome association and fragmentation upon completion of homologous pairing and synapsis. These aberrant chromosome associations were independent of OsDMC1. We found that classical nonhomologous end-joining mediated by Ku70 accounted for most of the ectopic associations in Osrad1. In addition, OsRAD1 interacts directly with OsHUS1 and OsRAD9, suggesting that these proteins act as a complex to promote DSB repair during rice meiosis. Together, these findings suggest that the 9-1-1 complex facilitates accurate meiotic recombination by suppressing nonhomologous end-joining during meiosis in rice.Meiosis comprises two successive cell divisions after a single S phase, generating four haploid products. To ensure proper chromosome segregation at the first meiotic division, crossovers (COs) are formed between homologous chromosomes (Kleckner, 2006). CO formation requires faithful repair of programmed DNA double-strand breaks (DSBs) introduced by the protein SPO11 (Keeney et al., 1997; Shinohara et al., 1997).Mitotic cells employ two basic strategies for DSB repair: homologous recombination (HR) and classical nonhomologous end-joining (C-NHEJ; Deriano and Roth, 2013). HR requires an undamaged template sequence for repair, while the C-NHEJ pathway involves direct ligation of the broken ends in a Ku-dependent manner. Both HR and C-NHEJ safeguard genome integrity during mitosis (Ceccaldi et al., 2016; Symington and Gautier, 2011). However, DSBs are preferentially repaired by HR during meiosis, because only this pathway generates COs. C-NHEJ competes with HR and creates de novo mutations in the gametes, indicating that this activity should be restricted during meiotic DSB repair. Previous studies have identified several factors essential for preventing C-NHEJ in meiosis (Goedecke et al., 1999; Martin et al., 2005; Adamo et al., 2010; Lemmens et al., 2013).Although the mechanism inhibiting C-NHEJ during meiosis is still elusive, regulators guaranteeing the success of the HR pathway have been extensively studied. Radiation sensitive1 (RAD1) is an evolutionarily conserved protein whose best-known function is checkpoint signaling. RAD1, a member of the ring-shaped RAD9-RAD1-HUS1 (9-1-1) complex, plays a crucial role in activating the pachytene checkpoint, a surveillance mechanism for monitoring the progression of meiotic HR in many organisms (Lydall et al., 1996; Hong and Roeder, 2002; Eichinger and Jentsch, 2010).In addition to their well-known roles in checkpoint signaling, members of 9-1-1 complex may also play a direct role in facilitating DSB repair and HR during meiosis. RAD1 is associated with both synapsed and unsynapsed chromosomes during prophase I in mouse (Freire et al., 1998). The homolog of RAD1 in Saccharomyces cerevisiae is Rad17, and rad17 mutant exhibits persistent Rad51 foci (Shinohara et al., 2003). Moreover, mutations in Rad17 lead to a reduced frequency of interhomolog recombination, aberrant synapsis, increased rates of ectopic recombination events, and illegitimate repair from the sister chromatids during meiosis (Grushcow et al., 1999). Recently, Rad17 was shown to be necessary for the efficient assembly of ZMM proteins (Shinohara et al., 2015). Apart from RAD1, the other partners of 9-1-1 were also shown to be involved in DSB repair. HUS1 is proved essential for meiotic DSB repair in Drosophila (Peretz et al., 2009). Moreover, Hus1 inactivation in mouse testicular germ cells results in persistent meiotic DNA damage, chromosomal defects, and germ cell depletion (Lyndaker et al., 2013). Nevertheless, little is known about the role of 9-1-1 proteins in higher plants. In Arabidopsis (Arabidopsis thaliana), mutants of RAD9 show increased sensitivity to genotoxic agents and delayed general repair of mitotic DSBs (Heitzeberg et al., 2004). A recent study indicates that HUS1 is involved in DSB repair of both mitotic and meiotic cells in rice (Che et al., 2014).In this study, we showed that OsRAD1 was required for the accurate repair of DSBs in rice during meiosis. Importantly, we demonstrated that the defective meiotic DSB repair in the Osrad1 mutants could be partially suppressed by blocking the C-NHEJ pathway. We also investigated the relationship between OsRAD1 and other key recombination proteins. Together, our findings indicated that the 9-1-1 complex plays a crucial role in the meiotic DSB repair mechanism.     

Suppression of BRCA1 sensitizes cells to proteasome inhibitors

BRCA1 is a multifunctional protein best known for its role in DNA repair and association with breast and ovarian cancers. To uncover novel biologically significant molecular functions of BRCA1, we tested a panel of 198 approved and experimental drugs to inhibit growth of MDA-MB-231 breast cancer cells depleted for BRCA1 by siRNA. 26S proteasome inhibitors bortezomib and carfilzomib emerged as a new class of selective BRCA1-targeting agents. The effect was confirmed in HeLa and U2OS cancer cell lines using two independent siRNAs, and in mouse embryonic stem (ES) cells with inducible deletion of Brca1. Bortezomib treatment did not cause any increase in nuclear foci containing phosphorylated histone H2AX, and knockdown of BRCA2 did not entail sensitivity to bortezomib, suggesting that the DNA repair function of BRCA1 may not be directly involved. We found that a toxic effect of bortezomib on BRCA1-depleted cells is mostly due to deregulated cell cycle checkpoints mediated by RB1-E2F pathway and 53BP1. Similar to BRCA1, depletion of RB1 also conferred sensitivity to bortezomib, whereas suppression of E2F1 or 53BP1 together with BRCA1 reduced induction of apoptosis after bortezomib treatment. A gene expression microarray study identified additional genes activated by bortezomib treatment only in the context of inactivation of BRCA1 including a critical involvement of the ERN1-mediated unfolded protein response. Our data indicate that BRCA1 has a novel molecular function affecting cell cycle checkpoints in a manner dependent on the 26S proteasome activity.BRCA1 is an important tumor suppressor gene whose germ-line or somatic inactivation is implicated in a significant number of breast and ovarian cancers.¹ Human BRCA1 encodes an 1863 amino-acid-long protein with a RING-finger domain at the N terminus and two BRCT domains located at the C terminus.^{2, 3} BRCT domains mediate interaction with phosphorylated proteins such as Abraxas, BACH1, CtIP and others involved in sensing DNA damage and assembly of the BRCA1-associated genome surveillance complex at sites of DNA breaks.⁴ The RING domain constitutively interacts with the BRCA1-associated RING domain protein (BARD1), forming a heterodimer having an E3 ubiquitin ligase activity.⁵ Ubiquitination of target proteins, including cell cycle or DNA repair-regulating proteins (e.g. CtIP (RBBP8), nucleophosmin (NPM1, B23), claspin (CLSPN) and others), occurs either at Lys48 residue of the ubiquitin leading to the 26S proteasome-mediated degradation of target proteins or at Lys6 or Lys63 having a trafficking and signaling role.⁶ A serine cluster coiled-coil domain spanning amino acids 1280–1524 contains multiple phosphorylation sites for ATM and ATR kinases activated by DNA damage.⁷ The same region also binds PALB2 protein linking BRCA1 to another major breast cancer predisposition gene BRCA2.⁸The most prominent function of BRCA1 is associated with its role in repair of DNA damage, particularly of double-stranded DNA breaks (DSBs), one of the most severe types of DNA lesions.⁹ BRCA1 is recruited to sites of DNA damage via a series of phosphorylation and ubiquitination events, where it serves as a binding scaffold for other DNA repair proteins,^{10, 11} ubiquitinates claspin, cyclin B and CDC25C, triggering cell cycle arrest to allow time for repair,¹² and facilitates BRCA2-mediated loading of RAD51 recombinase to enable the homologous recombination (HR) mechanism of DNA repair.⁹ In addition, BRCA1 may contribute to maintaining genome integrity by stabilizing the heterochromatin structure via ubiquitination of histone H2A.¹³ BRCA1 is also required for centrosome-dependent and -independent mitotic spindle formation, providing another route, by which loss of BRCA1 could promote chromosome instability and tumor formation.^{14, 15}Such a critical role of BRCA1 in DNA repair is exploited therapeutically. DNA-damaging agents, particularly DNA-crosslinking agents such as platinum-containing drugs, or ionizing radiation lead to the accumulation of DNA breaks requiring HR for repair and, therefore, are particularly toxic to BRCA1-deficient tumor cells.¹⁶ Pharmacological inhibitors of poly-(ADP-ribose) polymerases (PARPs) selectively kill BRCA1-deficient cells owing to defective HR, functioning as a back-up repair mechanism in the absence of the PARP-mediated repair of single-stranded DNA breaks.¹⁷ However, multiple mechanisms allow BRCA1-deficient cells to develop resistance to these drugs including elevated expression of the efflux transporters pumping the drugs out of the cell, secondary mutations restoring a functional BRCA1 protein and loss of 53BP1 protein, which counteracts BRCA1 and HR by blocking resection of DNA ends around the breaks (see Lord and Ashworth¹⁸ for the latest review). Therefore, additional efforts to identify small-molecule agents especially targeting BRCA1 functions unrelated to its DNA repair function are warranted.Here we performed a high-throughput chemical screen of BRCA1-depleted MDA-MB-231 cells using a collection of 198 US Food and Drug Administration (FDA)-approved and experimental drugs. We found that 26S proteasome inhibitors were more toxic to BRCA1 knockdown than control cells. Response of BRCA1-deficient cells to bortezomib involved deregulation of the RB1-mediated cell cycle checkpoint, activation of a noncanonical ERN1-mediated unfolded protein response and 53BP1-related G2/M cell cycle arrest. Our results reveal novel aspects of BRCA1 function unrelated to DNA repair.