Cell-type specific role of the RNA-binding protein, NONO, in the DNA double-strand break response in the mouse testes.
ABSTRACT: The tandem RNA recognition motif protein, NONO, was previously identified as a candidate DNA double-strand break (DSB) repair factor in a biochemical screen for proteins with end-joining stimulatory activity. Subsequent work showed that NONO and its binding partner, SFPQ, have many of the properties expected for bona fide repair factors in cell-based assays. Their contribution to the DNA damage response in intact tissue in vivo has not, however, been demonstrated. Here we compare DNA damage sensitivity in the testes of wild-type mice versus mice bearing a null allele of the NONO homologue (Nono gt). In wild-type mice, NONO protein was present in Sertoli, peritubular myoid, and interstitial cells, with an increase in expression following induction of DNA damage. As expected for the product of an X-linked gene, NONO was not detected in germ cells. The Nono gt/0 mice had at most a mild testis developmental phenotype in the absence of genotoxic stress. However, following irradiation at sublethal, 2-4 Gy doses, Nono gt/0 mice displayed a number of indicators of radiosensitivity as compared to their wild-type counterparts. These included higher levels of persistent DSB repair foci, increased numbers of apoptotic cells in the seminiferous tubules, and partial degeneration of the blood-testis barrier. There was also an almost complete loss of germ cells at later times following irradiation, evidently arising as an indirect effect reflecting loss of stromal support. Results demonstrate a role for NONO protein in protection against direct and indirect biological effects of ionizing radiation in the whole animal.
Project description:NONO, SFPQ and PSPC1 make up a family of proteins with diverse roles in transcription, RNA processing and DNA double-strand break (DSB) repair. To understand long-term effects of loss of NONO, we characterized murine embryonic fibroblasts (MEFs) from knockout mice. In the absence of genotoxic stress, wild-type and mutant MEFs showed similar growth rates and cell cycle distributions, and the mutants were only mildly radiosensitive. Further investigation showed that NONO deficiency led to upregulation of PSPC1, which replaced NONO in a stable complex with SFPQ. Knockdown of PSPC1 in a NONO-deficient background led to severe radiosensitivity and delayed resolution of DSB repair foci. The DNA-dependent protein kinase (DNA-PK) inhibitor, NU7741, sensitized wild-type and singly deficient MEFs, but had no additional effect on doubly deficient cells, suggesting that NONO/PSPC1 and DNA-PK function in the same pathway. We tested whether NONO and PSPC1 might also affect repair indirectly by influencing mRNA levels for other DSB repair genes. Of 12 genes tested, none were downregulated, and several were upregulated. Thus, NONO or related proteins are critical for DSB repair, NONO and PSPC1 are functional homologs with partially interchangeable functions and a compensatory response involving PSPC1 blunts the effect of NONO deficiency.
Project description:Noncycling and terminally differentiated (TD) cells display differences in radiosensitivity and DNA damage response. Unlike other TD cells, Sertoli cells express a mixture of proliferation inducers and inhibitors in vivo and can reenter the cell cycle. Being in a G1-like cell cycle stage, TD Sertoli cells are expected to repair DSBs by the error-prone nonhomologous end-joining pathway (NHEJ). Recently, we have provided evidence for the involvement of Ku-dependent NHEJ in protecting testis cells from DNA damage as indicated by persistent foci of the DNA double-strand break (DSB) repair proteins phospho-H2AX, 53BP1, and phospho-ATM in TD Sertoli cells of Ku70-deficient mice. Here, we analyzed the kinetics of 53BP1 foci induction and decay up to 12 h after 0.5 Gy gamma irradiation in DNA-PKcs-deficient (Prkdc scid ) and wild-type Sertoli cells. In nonirradiated mice and Prkdc scid Sertoli cells displayed persistent DSBs foci in around 12 % of cells and a fivefold increase in numbers of these DSB DNA damage-related foci relative to the wild type. In irradiated mice, Prkdc scid Sertoli cells showed elevated levels of DSB-indicating foci in 82 % of cells 12 h after ionizing radiation (IR) exposure, relative to 52 % of irradiated wild-type Sertoli cells. These data indicate that Sertoli cells respond to and repair IR-induced DSBs in vivo, with repair kinetics being slow in the wild type and inefficient in Prkdc scid . Applying the same dose of IR to Prdkc -/- and Ku -/- mouse embryonic fibroblast (MEF) cells revealed a delayed induction of 53BP1 DSB-indicating foci 5 min post-IR in Prdkc -/- cells. Inefficient DSB repair was evident 7 h post-IR in DNA-PKcs-deficient cells, but not in Ku -/- MEFs. Our data show that quiescent Sertoli cells repair genotoxic DSBs by DNA-PKcs-dependent NEHJ in vivo with a slower kinetics relative to somatic DNA-PKcs-deficient cells in vitro, while DNA-PKcs deficiency caused inefficient DSB repair at later time points post-IR in both conditions. These observations suggest that DNA-PKcs contributes to the fast and slow repair of DSBs by NHEJ.
Project description:Vertebrates express two A-type cyclins; both associate with and activate the CDK2 protein kinase. Cyclin A1 is required in the male germ line, but its molecular functions are incompletely understood. We observed specific induction of cyclin A1 expression and promoter activity after UV and gamma-irradiation which was mediated by p53. cyclin A1-/- cells showed increased radiosensitivity. To unravel a potential role of cyclin A1 in DNA repair, we performed a yeast triple hybrid screen and identified the Ku70 DNA repair protein as a binding partner and substrate of the cyclin A1-CDK2 complex. DNA double-strand break (DSB) repair was deficient in cyclin A1-/- cells. Further experiments indicated that A-type cyclins activate DNA DSB repair by mechanisms that depend on CDK2 activity and Ku proteins. Both cyclin A1 and cyclin A2 enhanced DSB repair by homologous recombination, but only cyclin A1 significantly activated nonhomologous end joining. DNA DSB repair was specific for A-type cyclins because cyclin E was ineffective. These findings establish a novel function for cyclin A1 and CDK2 in DNA DSB repair following radiation damage.
Project description:The model bryophyte Physcomitrella patens is unique among plants in supporting the generation of mutant alleles by facile homologous recombination-mediated gene targeting (GT). Reasoning that targeted transgene integration occurs through the capture of transforming DNA by the homology-dependent pathway for DNA double-strand break (DNA-DSB) repair, we analysed the genome-wide transcriptomic response to bleomycin-induced DNA damage and generated mutants in candidate DNA repair genes. Massively parallel (Illumina) cDNA sequencing identified potential participants in gene targeting. Transcripts encoding DNA repair proteins active in multiple repair pathways were significantly up-regulated. These included Rad51, CtIP, DNA ligase 1, Replication protein A and ATR in homology-dependent repair, Xrcc4, DNA ligase 4, Ku70 and Ku80 in non-homologous end-joining and Rad1, Tebichi/polymerase theta, PARP in microhomology-mediated end-joining. Differentially regulated cell-cycle components included up-regulated Rad9 and Hus1 DNA-damage-related checkpoint proteins and down-regulated D-type cyclins and B-type CDKs, commensurate with the imposition of a checkpoint at G2 of the cell cycle characteristic of homology-dependent DNA-DSB repair. Candidate genes, including ATP-dependent chromatin remodelling helicases associated with repair and recombination, were knocked out and analysed for growth defects, hypersensitivity to DNA damage and reduced GT efficiency. Targeted knockout of PpCtIP, a cell-cycle activated mediator of homology-dependent DSB resection, resulted in bleomycin-hypersensitivity and greatly reduced GT efficiency.
Project description:The inhibitory role of p53 in DNA double-strand break (DSB) repair seems contradictory to its tumor-suppressing property. The p53 isoform ?113p53/?133p53 is a p53 target gene that antagonizes p53 apoptotic activity. However, information on its functions in DNA damage repair is lacking. Here we report that ?113p53 expression is strongly induced by ?-irradiation, but not by UV-irradiation or heat shock treatment. Strikingly, ?113p53 promotes DNA DSB repair pathways, including homologous recombination, non-homologous end joining and single-strand annealing. To study the biological significance of ?113p53 in promoting DNA DSB repair, we generated a zebrafish ?113p53(M/M) mutant via the transcription activator-like effector nuclease technique and found that the mutant is more sensitive to ?-irradiation. The human ortholog, ?133p53, is also only induced by ?-irradiation and functions to promote DNA DSB repair. ?133p53-knockdown cells were arrested at the G2 phase at the later stage in response to ?-irradiation due to a high level of unrepaired DNA DSBs, which finally led to cell senescence. Furthermore, ?113p53/?133p53 promotes DNA DSB repair via upregulating the transcription of repair genes rad51, lig4 and rad52 by binding to a novel type of p53-responsive element in their promoters. Our results demonstrate that ?113p53/?133p53 is an evolutionally conserved pro-survival factor for DNA damage stress by preventing apoptosis and promoting DNA DSB repair to inhibit cell senescence. Our data also suggest that the induction of ?133p53 expression in normal cells or tissues provides an important tolerance marker for cancer patients to radiotherapy.
Project description:Technical improvements in clinical radiotherapy for maximizing cytotoxicity to the tumor while limiting negative impact on co-irradiated healthy tissues include the increasing use of particle therapy (e.g., proton therapy) worldwide. Yet potential differences in the biology of DNA damage induction and repair between irradiation with X-ray photons and protons remain elusive. We compared the differences in DNA double strand break (DSB) repair and survival of cells compromised in non-homologous end joining (NHEJ), homologous recombination repair (HRR) or both, after irradiation with an equal dose of X-ray photons, entrance plateau (EP) protons, and mid spread-out Bragg peak (SOBP) protons. We used super-resolution microscopy to investigate potential differences in spatial distribution of DNA damage foci upon irradiation. While DNA damage foci were equally distributed throughout the nucleus after X-ray photon irradiation, we observed more clustered DNA damage foci upon proton irradiation. Furthermore, deficiency in essential NHEJ proteins delayed DNA repair kinetics and sensitized cells to both, X-ray photon and proton irradiation, whereas deficiency in HRR proteins sensitized cells only to proton irradiation. We assume that NHEJ is indispensable for processing DNA DSB independent of the irradiation source, whereas the importance of HRR rises with increasing energy of applied irradiation.
Project description:The integrity of male germ cell genome is critical for the correct progression of spermatogenesis, successful fertilization, and proper development of the offspring. Several DNA repair pathways exist in male germ cells. However, unlike somatic cells, key components of such pathways remain largely unidentified. Gametogenetin (GGN) is a testis-enriched protein that has been shown to bind to the DNA repair protein FANCL via yeast-two-hybrid assays. This finding and its testis-enriched expression pattern raise the possibility that GGN plays a role in DNA repair during spermatogenesis. Herein we demonstrated that the largest isoform GGN1 interacted with components of DNA repair machinery in the mouse testis. In addition to FANCL, GGN1 interacted with the critical component of the Fanconi Anemia (FA) pathway FANCD2 and a downstream component of the BRCA pathway, BRCC36. To define the physiological function of GGN, we generated a Ggn null mouse line. A complete loss of GGN resulted in embryonic lethality at the very earliest period of pre-implantation development, with no viable blastocysts observed. This finding was consistent with the observation that Ggn mRNA was also expressed in lower levels in the oocyte and pre-implantation embryos. Moreover, pachytene spermatocytes of the Ggn heterozygous knockout mice showed an increased incidence of unrepaired DNA double strand breaks (DSBs). Together, our results suggest that GGN plays a role in male meiotic DSB repair and is absolutely required for the survival of pre-implantation embryos.
Project description: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.
Project description:Spermatogenesis is a complex process that generates haploid germ cells or spores and implements meiosis, a succession of two special cell divisions that are required for homologous chromosome segregation. During prophase to the first meiotic division, homologous recombination (HR) repairs Spo11-dependent DNA double-strand breaks (DSBs) in the presence of telomere movements to allow for chromosome pairing and segregation at the meiosis I division. In contrast to HR, non-homologous end joining (NHEJ), the major DSB repair mechanism during the G1 cell cycle phase, is downregulated during early meiotic prophase. At somatic mammalian telomeres, the NHEJ factor Ku70/80 inhibits HR, as does the Rap1 component of the shelterin complex. Here, we investigated the role of Ku70 and Rap1 in meiotic telomere redistribution and genome protection in spermatogenesis by studying single and double knockout mice. Ku70(-/-) mice display reduced testis size and compromised spermatogenesis, whereas meiotic telomere dynamics and chromosomal bouquet formation occurred normally in Ku70(-/-) and Ku70(-/-)Rap1(?/?) knockout spermatocytes. Elevated mid-preleptotene frequencies were associated with significantly increased DNA damage in Ku-deficient B spermatogonia, and in differentiated Sertoli cells. Significantly elevated levels of ?H2AX foci in Ku70(-/-) diplotene spermatocytes suggest compromised progression of DNA repair at a subset of DSBs. This might explain the elevated meiotic metaphase apoptosis that is present in Ku70-deficient stage XII testis tubules, indicating spindle assembly checkpoint activation. In summary, our data indicate that Ku70 is important for repairing DSBs in somatic cells and in late spermatocytes of the testis, thereby assuring the fidelity of spermatogenesis.
Project description:Fancj, the gene associated with Fanconi anemia (FA) Complementation Group J, encodes a DNA helicase involved in homologous recombination repair and the cellular response to replication stress. FANCJ functions in part through its interaction with key DNA repair proteins, including MutL homolog-1 (MLH1), Breast Cancer Associated gene-1 (BRCA1), and Bloom syndrome helicase (BLM). All three of these proteins are involved in a variety of events that ensure genome stability, including the events of DNA double strand break (DSB) repair during prophase I of meiosis. Meiotic DSBs are repaired through homologous recombination resulting in non-crossovers (NCO) or crossovers (CO). The frequency and placement of COs are stringently regulated to ensure that each chromosome receives at least one CO event, and that longer chromosomes receive at least one additional CO, thus facilitating the accurate segregation of homologous chromosomes at the first meiotic division. In the present study, we investigated the role of Fancj during prophase I using a gene trap mutant allele. Fancj (GT/GT) mutants are fertile, but their testes are very much smaller than wild-type littermates, predominantly as a result of impeded spermatogonial proliferation and mildly increased apoptosis during testis development in the fetus. This defect in spermatogonial proliferation is consistent with mutations in other FA genes. During prophase I, early events of synapsis and DSB induction/repair appear mostly normal in Fancj (GT/GT) males, and the FANCJ-interacting protein BRCA1 assembles normally on meiotic chromosome cores. However, MLH1 focus frequency is increased in Fancj (GT/GT) males, indicative of increased DSB repair via CO, and is concomitant with increased chiasmata at diakinesis. This increase in COs in the absence of FANCJ is associated with increased localization of BLM helicase protein, indicating that BLM may facilitate the increased rate of crossing over in Fancj (GT/GT) males. Taken together, these results demonstrate a critical role for FANCJ in spermatogenesis at two stages: firstly in the proliferative activity that gives rise to the full complement of testicular spermatogonia and secondly in the establishment of appropriate CO numbers during prophase I.