RFWD3-Mediated Ubiquitination Promotes Timely Removal of Both RPA and RAD51 from DNA Damage Sites to Facilitate Homologous Recombination.

RFWD3 is a recently identified Fanconi anemia protein FANCW whose E3 ligase activity toward RPA is essential in homologous recombination (HR) repair. However, how RPA ubiquitination promotes HR remained unknown. Here, we identified RAD51, the central HR protein, as another target of RFWD3. We show that RFWD3 polyubiquitinates both RPA and RAD51 in vitro and in vivo. Phosphorylation by ATR and ATM kinases is required for this activity in vivo. RFWD3 inhibits persistent mitomycin C (MMC)-induced RAD51 and RPA foci by promoting VCP/p97-mediated protein dynamics and subsequent degradation. Furthermore, MMC-induced chromatin loading of MCM8 and RAD54 is defective in cells with inactivated RFWD3 or expressing a ubiquitination-deficient mutant RAD51. Collectively, our data reveal a mechanism that facilitates timely removal of RPA and RAD51 from DNA damage sites, which is crucial for progression to the late-phase HR and suppression of the FA phenotype.

Genomic Instability and Cancer Risk Associated with Erroneous DNA Repair.

Many cancers develop as a consequence of genomic instability, which induces genomic rearrangements and nucleotide mutations. Failure to correct DNA damage in DNA repair defective cells, such as in BRCA1 and BRCA2 mutated backgrounds, is directly associated with increased cancer risk. Genomic rearrangement is generally a consequence of erroneous repair of DNA double-strand breaks (DSBs), though paradoxically, many cancers develop in the absence of DNA repair defects. DNA repair systems are essential for cell survival, and in cancers deficient in one repair pathway, other pathways can become upregulated. In this review, we examine the current literature on genomic alterations in cancer cells and the association between these alterations and DNA repair pathway inactivation and upregulation.

Mcm8 and Mcm9 form a complex that functions in homologous recombination repair induced by DNA interstrand crosslinks.

DNA interstrand crosslinks (ICLs) are highly toxic lesions that stall the replication fork to initiate the repair process during the S phase of vertebrates. Proteins involved in Fanconi anemia (FA), nucleotide excision repair (NER), and translesion synthesis (TS) collaboratively lead to homologous recombination (HR) repair. However, it is not understood how ICL-induced HR repair is carried out and completed. Here, we showed that the replicative helicase-related Mcm family of proteins, Mcm8 and Mcm9, forms a complex required for HR repair induced by ICLs. Chicken DT40 cells lacking MCM8 or MCM9 are viable but highly sensitive to ICL-inducing agents, and exhibit more chromosome aberrations in the presence of mitomycin C compared with wild-type cells. During ICL repair, Mcm8 and Mcm9 form nuclear foci that partly colocalize with Rad51. Mcm8-9 works downstream of the FA and BRCA2/Rad51 pathways, and is required for HR that promotes sister chromatid exchanges, probably as a hexameric ATPase/helicase.

Replication stress induces accumulation of FANCD2 at central region of large fragile genes.

During mild replication stress provoked by low dose aphidicolin (APH) treatment, the key Fanconi anemia protein FANCD2 accumulates on common fragile sites, observed as sister foci, and protects genome stability. To gain further insights into FANCD2 function and its regulatory mechanisms, we examined the genome-wide chromatin localization of FANCD2 in this setting by ChIP-seq analysis. We found that FANCD2 mostly accumulates in the central regions of a set of large transcribed genes that were extensively overlapped with known CFS. Consistent with previous studies, we found that this FANCD2 retention is R-loop-dependent. However, FANCD2 monoubiquitination and RPA foci formation were still induced in cells depleted of R-loops. Interestingly, we detected increased Proximal Ligation Assay dots between FANCD2 and R-loops following APH treatment, which was suppressed by transcriptional inhibition. Collectively, our data suggested that R-loops are required to retain FANCD2 in chromatin at the middle intronic region of large genes, while the replication stress-induced upstream events leading to the FA pathway activation are not triggered by R-loops.

FANCI-FANCD2 stabilizes the RAD51-DNA complex by binding RAD51 and protects the 5'-DNA end.

The FANCI-FANCD2 (I-D) complex is considered to work with RAD51 to protect the damaged DNA in the stalled replication fork. However, the means by which this DNA protection is accomplished have remained elusive. In the present study, we found that the I-D complex directly binds to RAD51, and stabilizes the RAD51-DNA filament. Unexpectedly, the DNA binding activity of FANCI, but not FANCD2, is explicitly required for the I-D complex-mediated RAD51-DNA filament stabilization. The RAD51 filament stabilized by the I-D complex actually protects the DNA end from nucleolytic degradation by an FA-associated nuclease, FAN1. This DNA end protection is not observed with the RAD51 mutant from FANCR patient cells. These results clearly answer the currently enigmatic question of how RAD51 functions with the I-D complex to prevent genomic instability at the stalled replication fork.

Histone chaperone activity of Fanconi anemia proteins, FANCD2 and FANCI, is required for DNA crosslink repair.

Fanconi anaemia (FA) is a rare hereditary disorder characterized by genomic instability and cancer susceptibility. A key FA protein, FANCD2, is targeted to chromatin with its partner, FANCI, and plays a critical role in DNA crosslink repair. However, the molecular function of chromatin-bound FANCD2-FANCI is still poorly understood. In the present study, we found that FANCD2 possesses nucleosome-assembly activity in vitro. The mobility of histone H3 was reduced in FANCD2-knockdown cells following treatment with an interstrand DNA crosslinker, mitomycin C. Furthermore, cells harbouring FANCD2 mutations that were defective in nucleosome assembly displayed impaired survival upon cisplatin treatment. Although FANCI by itself lacked nucleosome-assembly activity, it significantly stimulated FANCD2-mediated nucleosome assembly. These observations suggest that FANCD2-FANCI may regulate chromatin dynamics during DNA repair.

Tumor suppressor RecQL5 controls recombination induced by DNA crosslinking agents.

RecQ family DNA helicases function in the maintenance of genome stability. Mice deficient in RecQL5, one of five RecQ helicases, show a cancer predisposition phenotype, suggesting that RecQL5 plays a tumor suppressor role. RecQL5 interacts with Rad51, a key factor in homologous recombination (HR), and displaces Rad51 from Rad51-single stranded DNA (ssDNA) filaments in vitro. However, the precise roles of RecQL5 in the cell remain elusive. Here, we present evidence suggesting that RecQL5 is involved in DNA interstrand crosslink (ICL) repair. Chicken DT40 RECQL5 gene knockout (KO) cells showed sensitivity to ICL-inducing agents such as cisplatin (CDDP) and mitomycin C (MMC) and a higher number of chromosome aberrations in the presence of MMC than wild-type cells. The phenotypes of RECQL5 KO cells resembled those of Fanconi anemia gene KO cells. Genetic analysis using corresponding gene knockout cells showed that RecQL5 is involved in the FANCD1 (BRCA2)-dependent ICL repair pathway in which Rad51-ssDNA filament formation is promoted by BRCA2. The disappearance but not appearance of Rad51-foci was delayed in RECQL5 KO cells after MMC treatment. Deletion of Rad54, which processes the Rad51-ssDNA filament in HR, in RECQL5 KO cells increased sensitivity to CDDP and further delayed the disappearance of Rad51-foci, suggesting that RecQL5 and Rad54 have different effects on the Rad51-ssDNA filament. Furthermore, the frequency and variation of CDDP-induced gene conversion at the immunoglobulin locus were increased in RECQL5 KO cells. These results suggest that RecQL5 plays a role in regulating the incidence and quality of ICL-induced recombination.

A novel interplay between the Fanconi anemia core complex and ATR-ATRIP kinase during DNA cross-link repair.

When DNA replication is stalled at sites of DNA damage, a cascade of responses is activated in the cell to halt cell cycle progression and promote DNA repair. A pathway initiated by the kinase Ataxia teleangiectasia and Rad3 related (ATR) and its partner ATR interacting protein (ATRIP) plays an important role in this response. The Fanconi anemia (FA) pathway is also activated following genomic stress, and defects in this pathway cause a cancer-prone hematologic disorder in humans. Little is known about how these two pathways are coordinated. We report here that following cellular exposure to DNA cross-linking damage, the FA core complex enhances binding and localization of ATRIP within damaged chromatin. In cells lacking the core complex, ATR-mediated phosphorylation of two functional response targets, ATRIP and FANCI, is defective. We also provide evidence that the canonical ATR activation pathway involving RAD17 and TOPBP1 is largely dispensable for the FA pathway activation. Indeed DT40 mutant cells lacking both RAD17 and FANCD2 were synergistically more sensitive to cisplatin compared with either single mutant. Collectively, these data reveal new aspects of the interplay between regulation of ATR-ATRIP kinase and activation of the FA pathway.

DNA robustly stimulates FANCD2 monoubiquitylation in the complex with FANCI.

FANCI and FANCD2 form a complex, and play essential roles in the repair of interstrand DNA crosslinks (ICLs) by the Fanconi anemia (FA) pathway. FANCD2 is monoubiquitylated by the FA core complex, composed of 10 FA proteins including FANCL as the catalytic E3 subunit. FANCD2 monoubiquitylation can be reconstituted with purified minimal components, such as FANCI, E1, UBE2T (E2) and FANCL (E3) in vitro; however, its efficiency is quite low as compared to the in vivo monoubiquitylation of FANCD2. In this study, we found that various forms of DNA, such as single-stranded, double-stranded and branched DNA, robustly stimulated the FANCD2 monoubiquitylation in vitro up to a level comparable to its in vivo monoubiquitylation. This stimulation of the FANCD2 monoubiquitylation strictly required FANCI, suggesting that FANCD2 monoubiquitylation may occur in the FANCI-FANCD2 complex. A FANCI mutant that was defective in DNA binding was also significantly defective in FANCD2 monoubiquitylation in vitro. In the presence of 5' flapped DNA, a DNA substrate mimicking the arrested replication fork, about 70% of the input FANCD2 was monoubiquitylated, while less than 1% FANCD2 monoubiquitylation was observed in the absence of the DNA. Therefore, DNA may be the unidentified factor required for proper FANCD2 monoubiquitylation.

Predisposition to cancer caused by genetic and functional defects of mammalian Atad5.

ATAD5, the human ortholog of yeast Elg1, plays a role in PCNA deubiquitination. Since PCNA modification is important to regulate DNA damage bypass, ATAD5 may be important for suppression of genomic instability in mammals in vivo. To test this hypothesis, we generated heterozygous (Atad5(+/m)) mice that were haploinsuffficient for Atad5. Atad5(+/m) mice displayed high levels of genomic instability in vivo, and Atad5(+/m) mouse embryonic fibroblasts (MEFs) exhibited molecular defects in PCNA deubiquitination in response to DNA damage, as well as DNA damage hypersensitivity and high levels of genomic instability, apoptosis, and aneuploidy. Importantly, 90% of haploinsufficient Atad5(+/m) mice developed tumors, including sarcomas, carcinomas, and adenocarcinomas, between 11 and 20 months of age. High levels of genomic alterations were evident in tumors that arose in the Atad5(+/m) mice. Consistent with a role for Atad5 in suppressing tumorigenesis, we also identified somatic mutations of ATAD5 in 4.6% of sporadic human endometrial tumors, including two nonsense mutations that resulted in loss of proper ATAD5 function. Taken together, our findings indicate that loss-of-function mutations in mammalian Atad5 are sufficient to cause genomic instability and tumorigenesis.

APTX acts in DNA double-strand break repair in a manner distinct from XRCC4.

Aprataxin (APTX), the product of the causative gene for hereditary neurogenerative syndromes Ataxia-oculomotor apraxia 1 and early onset ataxia with oculomotor apraxia and hypoalbuminemia, has an enzymatic activity of removing adenosine monophosphate from DNA 5'-end, which arises from abortive ligation by DNA ligases. It is also reported that APTX physically binds to XRCC1 and XRCC4, suggesting its involvement in DNA single-strand break repair (SSBR) and DNA double-strand break repair (DSBR) via non-homologous end joining pathway. Although the involvement of APTX in SSBR in association with XRCC1 has been established, the significance of APTX in DSBR and its interaction with XRCC4 have remained unclear. Here, we generated APTX knock-out (APTX-/-) cell from human osteosarcoma U2OS through CRISPR/Cas9-mediated genome editing system. APTX-/- cells exhibited increased sensitivity toward ionizing radiation (IR) and Camptothecin in association with retarded DSBR, as shown by increased number of retained γH2AX foci. However, the number of retained 53BP1 foci in APTX-/- cell was not discernibly different from wild-type cells, in stark contrast to XRCC4-depleted cells. The recruitment of GFP-tagged APTX (GFP-APTX) to the DNA damage sites was examined by laser micro-irradiation and live-cell imaging analysis using confocal microscope. The accumulation of GFP-APTX on the laser track was attenuated by siRNA-mediated depletion of XRCC1, but not XRCC4. Moreover, the deprivation of APTX and XRCC4 displayed additive inhibitory effects on DSBR after IR exposure and end joining of GFP reporter. These findings collectively suggest that APTX acts in DSBR in a manner distinct from XRCC4.

Proteomic Characterization of SAS Cell-Derived Extracellular Vesicles in Relation to Both BPA and Neutron Irradiation Doses.

Boron neutron capture therapy (BNCT) is a selective radiotherapy based on nuclear reaction that occurs when 10B atoms accumulated in cancer cells are irradiated by thermal neutrons, triggering a nuclear fission response leading to cell death. Despite its growing importance in cancer treatment, molecular characterization of its effects is still lacking. In this context, proteomics investigation can be useful to study BNCT effect and identify potential biomarkers. Hence, we performed proteomic analysis with nanoLC-MS/MS (liquid chromatography coupled to tandem mass spectrometry) on extracellular vesicles (EVs) isolated from SAS cultures treated or not with 10B-boronophenylalanine (BPA) and different doses of neutron irradiation, to study the cellular response related to both boron administration and neutrons action. Despite the interference of fetal bovine serum in the medium, we were able to stratify BPA- and BPA+ conditions and to identify EVs-derived proteins characterizing pathways potentially related to a BNCT effect such as apoptosis, DNA repair and inflammatory response. In particular, KLF11, SERPINA1 and SERPINF2 were up-regulated in BPA+, while POLE and SERPINC1 were up-regulated in BPA-. These results provide the first proteomic investigation of EVs treated with BNCT in different conditions and highlight the potentiality of proteomics for improving biomarkers identification and mechanisms understanding of BNCT.

Relative biological effectiveness for epithermal neutron beam contaminated with fast neutrons in the linear accelerator-based boron neutron capture therapy system coupled to a solid-state lithium target.

This study aimed to quantify the relative biological effectiveness (RBE) for epithermal neutron beam contaminated with fast neutrons in the accelerator-based boron neutron capture therapy (BNCT) system coupled to a solid-state lithium target. The experiments were performed in National Cancer Center Hospital (NCCH), Tokyo, Japan. Neutron irradiation with the system provided by Cancer Intelligence Care Systems (CICS), Inc. was performed. X-ray irradiation, which was assigned as the reference group, was also performed using a medical linear accelerator (LINAC) equipped in NCCH. The four cell lines (SAS, SCCVII, U87-MG and NB1RGB) were utilized to quantify RBE value for the neutron beam. Before both of those irradiations, all cells were collected and dispensed into vials. The doses of 10% cell surviving fraction (SF) (D10) were calculated by LQ model fitting. All cell experiments were conducted in triplicate at least. Because the system provides not only neutrons, but gamma-rays, the contribution from the gamma-rays to the survival fraction were subtracted in this study. D10 value of SAS, SCCVII, U87-MG and NB1RGB for the neutron beam was 4.26, 4.08, 5.81 and 2.72 Gy, respectively, while that acquired by the X-ray irradiation was 6.34, 7.21, 7.12 and 5.49 Gy, respectively. Comparison of both of the D10 values, RBE value of SAS, SCCVII, U87-MG and NB1RGB for the neutron beam was calculated as 1.7, 2.2, 1.3 and 2.5, respectively, and the average RBE value was 1.9. This study investigated RBE of the epithermal neutron beam contaminated with fast neutrons in the accelerator-based BNCT system coupled to a solid-state lithium target.

The role of SNM1 family nucleases in etoposide-induced apoptosis.

DNA double strand breaks (DSBs) induced by etoposide, an inhibitor of DNA topoisomerase II, are repaired mainly by non-homologous end joining (NHEJ). Unexpectedly, it was found that at high doses of etoposide, proteins involved in NHEJ, such as KU70/80, DNA-PKcs and ARTEMIS/SNM1C, trigger apoptosis rather than repair of DSBs. Because ARTEMIS is a member of the SNM1 protein family that includes SNM1A and APOLLO/SNM1B, this study examined whether SNM1A and/or APOLLO are also involved in etoposide-induced apoptosis. Using SNM1A(-/-) and APOLLO(-/-) cells, it was found that both SNM1A and APOLLO participate in etoposide-induced apoptosis. Although cell viability monitored by MTT assay did not differ between SNM1A(-/-)/APOLLO(-/-)/ARTEMIS(-/-), SNM1A(-/-)/APOLLO(-/-), and single gene knockout cells, DNA fragmentation monitored by TUNEL assay differed between these cells, suggesting that the three SNM1 family nucleases function independently, at least during the induction of apoptotic DNA fragmentation.

Regulation of the Fanconi Anemia DNA Repair Pathway by Phosphorylation and Monoubiquitination.

The Fanconi anemia (FA) DNA repair pathway coordinates a faithful repair mechanism for stalled DNA replication forks caused by factors such as DNA interstrand crosslinks (ICLs) or replication stress. An important role of FA pathway activation is initiated by monoubiquitination of FANCD2 and its binding partner of FANCI, which is regulated by the ATM-related kinase, ATR. Therefore, regulation of the FA pathway is a good example of the contribution of ATR to genome stability. In this short review, we summarize the knowledge accumulated over the years regarding how the FA pathway is activated via phosphorylation and monoubiquitination.

The FHA domain of PNKP is essential for its recruitment to DNA damage sites and maintenance of genome stability.

Polynucleotide kinase phosphatase (PNKP) has dual enzymatic activities as kinase and phosphatase for DNA ends, which are the prerequisite for the ligation, and thus is involved in base excision repair, single-strand break repair and non-homologous end joining for double-strand break (DSB) repair. In this study, we examined mechanisms for the recruitment of PNKP to DNA damage sites by laser micro-irradiation and live-cell imaging analysis using confocal microscope. We show that the forkhead-associated (FHA) domain of PNKP is essential for the recruitment of PNKP to DNA damage sites. Arg35 and Arg48 within the FHA domain are required for interactions with XRCC1 and XRCC4. PNKP R35A/R48A mutant failed to accumulate on the laser track and siRNA-mediated depletion of XRCC1 and/or XRCC4 reduced PNKP accumulation on the laser track, indicating that PNKP is recruited to DNA damage sites via the interactions between its FHA domain and XRCC1 or XRCC4. Furthermore, cells expressing PNKP R35A/R48A mutant exhibited increased sensitivity toward ionizing radiation in association with delayed SSB and DSB repair and genome instability, represented by micronuclei and chromosome bridges. Taken together, these findings revealed the importance of PNKP recruitment to DNA damage sites via its FHA domain for DNA repair and maintenance of genome stability.

Vav3 modulates B cell receptor responses by regulating phosphoinositide 3-kinase activation.

To elucidate the mechanism(s) by which Vav3, a new member of the Vav family proteins, participates in B cell antigen receptor (BCR) signaling, we have generated a B cell line deficient in Vav3. Here we report that Vav3 influences phosphoinositide 3-kinase (PI3K) function through Rac1 in that phosphatidylinositol-3,4,5-trisphosphate (PIP3) generation was attenuated by loss of Vav3 or by expression of a dominant negative form of Rac1. The functional interaction between PI3K and Rac1 was also demonstrated by increased PI3K activity in the presence of GTP-bound Rac1. In addition, we show that defects of calcium mobilization and c-Jun NH2-terminal kinase (JNK) activation in Vav3-deficient cells are relieved by deletion of a PIP3 hydrolyzing enzyme, SH2 domain-containing inositol polyphosphate 5'-phosphatase (SHIP). Hence, our results suggest a role for Vav3 in regulating the B cell responses by promoting the sustained production of PIP3 and thereby calcium flux.

Bacterial SOS Genes mucAB/umuDC Promote Mouse Tumors by Activating Oncogenes Nedd9/Aurkb via a miR-145 Sponge.

The mechanism of cancer induction involves an aberrant expression of oncogenes whose functions can be controlled by RNAi with miRNA. Even foreign bacterial RNA may interfere with the expression of oncogenes. Here we show that bacterial plasmid mucAB and its Escherichia coli genomic homolog umuDC, carrying homologies that match the mouse anti-miR-145, sequestered the miR-145 function in mouse BALB 3T3 cells in a tetracycline (Tet)-inducible manner, activated oncogene Nedd9 and its downstream Aurkb, and further enhanced microcolony formation and cellular transformation as well as the short fragments of the bacterial gene containing the anti-miR-145 sequence. Furthermore, mucAB transgenic mice showed a 1.7-fold elevated tumor incidence compared with wild-type mice after treatments with 3-methylcolanthrene. However, the mutation frequency in intestinal stem cells of the mucAB transgenic mice was unchanged after treatment with X-rays or ethyl-nitrosourea, indicating that the target of mucAB/umuDC is the promotion stage in carcinogenesis. IMPLICATIONS: Foreign bacterial genes can exert oncogenic activity via RNAi, if endogenously expressed. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/18/9/1271/F1.large.jpg.

USP42 enhances homologous recombination repair by promoting R-loop resolution with a DNA-RNA helicase DHX9.

The nucleus of mammalian cells is compartmentalized by nuclear bodies such as nuclear speckles, however, involvement of nuclear bodies, especially nuclear speckles, in DNA repair has not been actively investigated. Here, our focused screen for nuclear speckle factors involved in homologous recombination (HR), which is a faithful DNA double-strand break (DSB) repair mechanism, identified transcription-related nuclear speckle factors as potential HR regulators. Among the top hits, we provide evidence showing that USP42, which is a hitherto unidentified nuclear speckles protein, promotes HR by facilitating BRCA1 recruitment to DSB sites and DNA-end resection. We further showed that USP42 localization to nuclear speckles is required for efficient HR. Furthermore, we established that USP42 interacts with DHX9, which possesses DNA-RNA helicase activity, and is required for efficient resolution of DSB-induced R-loop. In conclusion, our data propose a model in which USP42 facilitates BRCA1 loading to DSB sites, resolution of DSB-induced R-loop and preferential DSB repair by HR, indicating the importance of nuclear speckle-mediated regulation of DSB repair.