研究者詳細 - 柴田　淳史

論文 - 柴田　淳史

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DNA Double-Strand Break Resection Occurs during Non-homologous End Joining in G1 but Is Distinct from Resection during Homologous Recombination

Ronja Biehs, Monika Steinlage, Olivia Barton, Szilvia Juhasz, Julia Kuenzel, Julian Spies, Atsushi Shibata, Penny A. Jeggo, Markus Loebrich

MOLECULAR CELL （CELL PRESS） 65 671 - + 2017年02月

研究論文（学術雑誌）, 共著, 責任著者, 査読有り, ISSN 1097-2765

Canonical non-homologous end joining (c-NHEJ) repairs DNA double-strand breaks (DSBs) in G1 cells with biphasic kinetics. We show that DSBs repaired with slow kinetics, including those localizing to heterochromatic regions or harboring additional lesions at the DSB site, undergo resection prior to repair by c-NHEJ and not alt-NHEJ. Resection-dependent c-NHEJ represents an inducible process during which Plk3 phosphorylates CtIP, mediating its interaction with Brca1 and promoting the initiation of resection. Mre11 exonuclease, EXD2, and Exo1 execute resection, and Artemis endonuclease functions to complete the process. If resection does not commence, then repair can ensue by c-NHEJ, but when executed, Artemis is essential to complete resection-dependent c-NHEJ. Additionally, Mre11 endonuclease activity is dispensable for resection in G1. Thus, resection in G1 differs from the process in G2 that leads to homologous recombination. Resection-dependent c-NHEJ significantly contributes to the formation of deletions and translocations in G1, which represent important initiating events in carcinogenesis.

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Regulation of pairing between broken DNA-containing chromatin regions by Ku80, DNA-PKcs, ATM, and 53BP1

Motohiro Yamauchi, Atsushi Shibata, Keiji Suzuki, Masatoshi Suzuki, Atsuko Niimi, Hisayoshi Kondo, Miwa Miura, Miyako Hirakawa, Keiko Tsujita, Shunichi Yamashita, Naoki Matsuda

SCIENTIFIC REPORTS （NATURE PUBLISHING GROUP） 7 41812 2017年02月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 2045-2322

Chromosome rearrangement is clinically and physiologically important because it can produce oncogenic fusion genes. Chromosome rearrangement requires DNA double-strand breaks (DSBs) at two genomic locations and misrejoining between the DSBs. Before DSB misrejoining, two DSB-containing chromatin regions move and pair with each other; however, the molecular mechanism underlying this process is largely unknown. We performed a spatiotemporal analysis of ionizing radiation-induced foci of p53-binding protein 1 (53BP1), a marker for DSB-containing chromatin. We found that some 53BP1 foci were paired, indicating that the two damaged chromatin regions neighboured one another. We searched for factors regulating the foci pairing and found that the number of paired foci increased when Ku80, DNA-PKcs, or ATM was absent. In contrast, 53BP1 depletion reduced the number of paired foci and dicentric chromosomes-an interchromosomal rearrangement. Foci were paired more frequently in heterochromatin than in euchromatin in control cells. Additionally, the reduced foci pairing in 53BP1-depleted cells was rescued by concomitant depletion of a heterochromatin building factor such as kRPP ASSOCIATED box-associated protein 1 or chromodomain helicase DNA-binding protein 3. These findings indicate that pairing between DSB-containing chromatin regions was suppressed by Ku80, DNA-PKcs, and ATM, and this pairing was promoted by 53BP1 through chromatin relaxation.

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Mitotic catastrophe is a putative mechanism underlying the weak correlation between sensitivity to carbon ions and cisplatin

Daijiro Kobayashi, Takahiro Oike, Atsushi Shibata, Atsuko Niimi, Yoshiki Kubota, Makoto Sakai, Napapat Amornwhichet, Yuya Yoshimoto, Yoshihiko Hagiwara, Yuka Kimura, Yuka Hirota, Hiro Sato, Mayu Isono, Yukari Yoshida, Takashi Kohno, Tatsuya Ohno, Takashi Nakano

SCIENTIFIC REPORTS （NATURE PUBLISHING GROUP） 7 40588 2017年01月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 2045-2322

In cancer therapy today, carbon ion radiotherapy is used mainly as monotherapy, whereas cisplatin is used concomitantly with X-ray radiotherapy. The effectiveness of concomitant carbon ions and cisplatin is unclear. To obtain the information on the mechanisms potentially shared between carbon ions or X-rays and cisplatin, we assessed the correlation of sensitivity to the single treatments. In 20 human cancer cell lines, sensitivity to X-rays strongly correlated with sensitivity to cisplatin, indicating the presence of potentially shared target mechanisms. Interestingly, the correlation of sensitivity to carbon ions and cisplatin was much weaker than that of sensitivity to X-rays and cisplatin, indicating the presence of potentially different target mechanisms between carbon ions and cisplatin. Assessment of clonogenic cell death by 4',6-diamidino-2-phenylindole dihydrochloride staining showed that mitotic catastrophe was more efficiently induced by carbon ions than by the same physical dose of X-rays, while apoptosis and senescence were not. These data indicate that the correlation of sensitivity to carbon ions and cisplatin is weaker than that of sensitivity to X-rays and cisplatin, which are helpful as biological basis to understand the potentially shared mechanism among these treatments. Further investigation is mandatory to elucidate the clinical efficacy of carbon ions and cisplatin combination.

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BRCA1 Directs the Repair Pathway to Homologous Recombination by Promoting 53BP1 Dephosphorylation

Mayu Isono, Atsuko Niimi, Takahiro Oike, Yoshihiko Hagiwara, Hiro Sato, Ryota Sekine, Yukari Yoshida, Shin-Ya Isobe, Chikashi Obuse, Ryotaro Nishi, Elena Petricci, Shinichiro Nakada, Takashi Nakano, Atsushi Shibata

CELL REPORTS （CELL PRESS） 18 （ 2 ） 520 - 532 2017年01月

研究論文（学術雑誌）, 共著, 最終著者, 責任著者, 査読有り, ISSN 2211-1247

BRCA1 promoteshomologous recombination (HR) by activating DNA-end resection. By contrast, 53BP1 forms a barrier that inhibits DNA-end resection. Here, we show that BRCA1 promotes DNA-end resection by relieving the 53BP1-dependent barrier. We show that 53BP1 is phosphorylated by ATM in S/G(2) phase, promoting RIF1 recruitment, which inhibits resection. 53BP1 is promptly dephosphorylated and RIF1 released, despite remaining unrepaired DNA double-strand breaks (DSBs). When resection is impaired by CtIP/MRE11 endonuclease inhibition, 53BP1 phosphorylation and RIF1 are sustained due to ongoing ATM signaling. BRCA1 depletion also sustains 53BP1 phosphorylation and RIF1 recruitment. We identify the phosphatase PP4C as having a major role in 53BP1 dephosphorylation and RIF1 release. BRCA1 or PP4C depletion impairs 53BP1 repositioning, EXO1 recruitment, and HR progression. 53BP1 or RIF1 depletion restores resection, RAD51 loading, and HR in PP4C-depleted cells. Our findings suggest that BRCA1 promotes PP4C-dependent 53BP1 dephosphorylation and RIF1 release, directing repair toward HR.

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Identification of DNA Double Strand Breaks at Chromosome Boundaries Along the Track of Particle Irradiation

Atsuko Niimi, Motohiro Yamauchi, Siripan Limsirichaikul, Ryota Sekine, Takahiro Oike, Hiro Sato, Keiji Suzuki, Kathryn D. Held, Takashi Nakano, Atsushi Shibata

GENES CHROMOSOMES & CANCER （Wiley Periodicals, Inc.） 55 （ 8 ） 650 - 660 2016年04月

研究論文（学術雑誌）, 共著, 最終著者, 責任著者, 査読有り, ISSN 1045-2257

Chromosomal translocations arise from misrejoining of DNA double strand breaks (DSBs) between loci located on two chromosomes. One current model suggests that spatial proximity of potential chromosomal translocation partners influences translocation probability. Ionizing radiation (IR) is a potent inducer of translocations. Accumulating evidence demonstrates that particle irradiation more frequently causes translocations compared with X-ray irradiation. This observation has led to the hypothesis that the high frequency of translocations after particle irradiation may be due to the formation of DSBs at chromosome boundaries along the particle track, because such DSBs can be misrejoined between distinct chromosomes. In this study, we simultaneously visualized the site of IR-induced DSBs and chromosome position by combining Immunofluorescence and fluorescence in situ hybridization. Importantly, the frequency of gamma H2AX foci at the chromosome boundary of chromosome 1 after carbon-ion irradiation was >4-fold higher than that after X-ray irradiation. This observation is consistent with the idea that particle irradiation generates DSBs at the boundaries of two chromosomes along the track. Further, we showed that resolution of gamma H2AX foci at chromosome boundaries is prevented by inhibition of DNA-PKcs activity, indicating that the DSB repair is NHEJ-dependent. Finally, we found that gamma H2AX foci at chromosome boundaries after carbon-ion irradiation contain DSBs undergoing DNA-end resection, which promotes repair utilizing microhomology mediated end-joining during translocation. Taken together, our study suggests that the frequency of DSB formation at chromosome boundaries is associated with the incidence of chromosomal translocations, supporting the notion that the spatial proximity between breaks is an important factor in translocation formation. (C) 2016 Wiley Periodicals, Inc.

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Visualization of complex DNA double-strand breaks in a tumor treated with carbon ion radiotherapy

Takahiro Oike, Atsuko Niimi, Noriyuki Okonogi, Kazutoshi Murata, Akihiko Matsumura, Shin-Ei Noda, Daijiro Kobayashi, Mototaro Iwanaga, Keisuke Tsuchida, Tatsuaki Kanai, Tatsuya Ohno, Atsushi Shibata, Takashi Nakano

SCIENTIFIC REPORTS （NATURE PUBLISHING GROUP） 6 22275 2016年03月

研究論文（学術雑誌）, 共著, 責任著者, 査読有り, ISSN 2045-2322

Carbon ion radiotherapy shows great potential as a cure for X-ray-resistant tumors. Basic research suggests that the strong cell-killing effect induced by carbon ions is based on their ability to cause complex DNA double-strand breaks (DSBs). However, evidence supporting the formation of complex DSBs in actual patients is lacking. Here, we used advanced high-resolution microscopy with deconvolution to show that complex DSBs are formed in a human tumor clinically treated with carbon ion radiotherapy, but not in a tumor treated with X-ray radiotherapy. Furthermore, analysis using a physics model suggested that the complexity of radiotherapy-induced DSBs is related to linear energy transfer, which is much higher for carbon ion beams than for X-rays. Visualization of complex DSBs in clinical specimens will help us to understand the anti-tumor effects of carbon ion radiotherapy.

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RAD51 and BRCA2 Enhance Oncolytic Adenovirus Type 5 Activity in Ovarian Cancer

Laura A. Tookman, Ashley K. Browne, Claire M. Connell, Gemma Bridge, Carin K. Ingemarsdotter, Suzanne Dowson, Atsushi Shibata, Michelle Lockley, Sarah A. Martin, Iain A. McNeish

MOLECULAR CANCER RESEARCH （AMER ASSOC CANCER RESEARCH） 14 （ 1 ） 44 - 55 2016年01月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 1541-7786

Homologous recombination (HR) function is critically important in high-grade serous ovarian cancer (HGSOC). HGSOC with intact HR has a worse prognosis and is less likely to respond to platinum chemotherapy and PARP inhibitors. Oncolytic adenovirus, a novel therapy for human malignancies, stimulates a potent DNA damage response that influences overall antitumor activity. Here, the importance of HR was investigated by determining the efficacy of adenovirus type 5 (Ad5) vectors in ovarian cancer. Using matched BRCA2-mutant and wild-type HGSOC cells, it was demonstrated that intact HR function promotes viral DNA replication and augments overall efficacy, without influencing viral DNA processing. These data were confirmed in a wider panel of HR competent and defective ovarian cancer lines. Mechanistically, both BRCA2 and RAD51 localize to viral replication centers within the infected cell nucleus and that RAD51 localization occurs independently of BRCA2. In addition, a direct interaction was identified between RAD51 and adenovirus E2 DNA binding protein. Finally, using functional assays of HR competence, despite inducing degradation of MRE11, Ad5 infection does not alter cellular ability to repair DNA double-strand break damage via HR. These data reveal that Ad5 redistributes critical HR components to viral replication centers and enhances cytotoxicity. (C)2015 AACR.

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SETDB1, HP1 and SUV39 promote repositioning of 53BP1 to extend resection during homologous recombination in G2 cells

Meryem Alagoz, Yoko Katsuki, Hideaki Ogiwara, Tomoo Ogi, Atsushi Shibata, Andreas Kakarougkas, Penny Jeggo

NUCLEIC ACIDS RESEARCH （OXFORD UNIV PRESS） 43 （ 16 ） 7931 - 7944 2015年09月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0305-1048

Recent studies have shown that homologous recombination ( HR) requires chromatin repression as well as relaxation at DNA double strand breaks ( DSBs). HP1 and SUV39H1/ 2 are repressive factors essential for HR. Here, we identify SETDB1 as an additional compacting factor promoting HR. Depletion of HP1, SUV39, SETDB1 or BRCA1 confer identical phenotypes. The repressive factors, like BRCA1, are dispensable for the initiation of resection but promote the extension step causing diminished RPA or RAD51 foci and HR in irradiated G2 cells. Depletion of the compacting factors does not inhibit BRCA1 recruitment but at 8 h post IR, BRCA1 foci are smaller and aberrantly positioned compared to control cells. BRCA1 promotes 53BP1 repositioning to the periphery of enlarged foci and formation of a devoid core with BRCA1 becoming enlarged and localized internally to 53BP1. Depletion of the compacting factors precludes these changes at irradiation- induced foci. Thus, the repressive factors are required for BRCA1 function in promoting the repositioning of 53BP1 during HR. Additionally, depletion of these repressive factors in undamaged cells causes diminished sister chromatid association at centromeric sequences. We propose a model for how these findings may be functionally linked.

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Carbon-ion beams effectively induce growth inhibition and apoptosis in human neural stem cells compared with glioblastoma A172 cells

Mayu Isono, Yukari Yoshida, Akihisa Takahashi, Takahiro Oike, Atsushi Shibata, Yoshiki Kubota, Tatsuaki Kanai, Tatsuya Ohno, Takashi Nakano

JOURNAL OF RADIATION RESEARCH （OXFORD UNIV PRESS） 56 （ 5 ） 856 - 861 2015年06月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0449-3060

Carbon-ion radiotherapy (CIRT) holds promise in the treatment of glioblastoma, an aggressive X-ray-resistant brain tumor. However, since glioblastoma cells show a highly invasive nature, carbon-ion (C-ion) irradiation of normal tissues surrounding the tumor is inevitable. Recent studies have revealed the existence of neural stem cells in the adult brain. Therefore, the damaging effect of C-ion beams on the neural stem cells has to be carefully considered in the treatment planning of CIRT. Here, we investigated the growth and death mode of human neural stem cells (hNSCs) and glioblastoma A172 cells after X-ray or C-ion beam irradiation. The X-ray dose resulting in a 50% growth rate (D-50) was 0.8 Gy in hNSCs and 3.0 Gy in A172 cells, while the D-50 for C-ion beams was 0.4 Gy in hNSCs and 1.6 Gy in A172 cells; the relative biological effectiveness value of C-ion beams was 2.0 in hNSCs and 1.9 in A172 cells. Importantly, both X-rays and C-ion beams preferentially induced apoptosis, not necrosis, in hNSCs; however, radiation-induced apoptosis was less evident in A172 cells. The apoptosis-susceptible nature of the irradiated hNSCs was associated with prolonged upregulation of phosphorylated p53, whereas the apoptosis-resistant nature of A172 cells was associated with a high basal level of nuclear factor kappa B expression. Taken together, these data indicate that apoptosis is the major cell death pathway in hNSCs after irradiation. The high sensitivity of hNSCs to C-ion beams underscores the importance of careful target volume delineation in the treatment planning of CIRT for glioblastoma.

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The EGFR mutation status affects the relative biological effectiveness of carbon-ion beams in non-small cell lung carcinoma cells

Napapat Amornwichet, Takahiro Oike, Atsushi Shibata, Chaitanya S. Nirodi, Hideaki Ogiwara, Haruhiko Makino, Yuka Kimura, Yuka Hirota, Mayu Isono, Yukari Yoshida, Tatsuya Ohno, Takashi Kohno, Takashi Nakano

SCIENTIFIC REPORTS （NATURE PUBLISHING GROUP） 5 11305 2015年06月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 2045-2322

Carbon-ion radiotherapy (CIRT) holds promise to treat inoperable locally-advanced non-small cell lung carcinoma (NSCLC), a disease poorly controlled by standard chemoradiotherapy using X-rays. Since CIRT is an extremely limited medical resource, selection of NSCLC patients likely to benefit from it is important; however, biological predictors of response to CIRT are ill-defined. The present study investigated the association between the mutational status of EGFR and KRAS, driver genes frequently mutated in NSCLC, and the relative biological effectiveness (RBE) of carbon-ion beams over X-rays. The assessment of 15 NSCLC lines of different EGFR/KRAS mutational status and that of isogenic NSCLC lines expressing wild-type or mutant EGFR revealed that EGFR-mutant NSCLC cells, but not KRAS-mutant cells, show low RBE. This was attributable to (i) the high X-ray sensitivity of EGFR-mutant cells, since EGFR mutation is associated with a defect in non-homologous end joining, a major pathway for DNA double-strand break (DSB) repair, and (ii) the strong cell-killing effect of carbon-ion beams due to poor repair of carbon-ion beam-induced DSBs regardless of EGFR mutation status. These data highlight the potential of EGFR mutation status as a predictor of response to CIRT, i. e., CIRT may show a high therapeutic index in EGFR mutation-negative NSCLC.

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Pre-Exposure to Ionizing Radiation Stimulates DNA Double Strand Break End Resection, Promoting the Use of Homologous Recombination Repair

Nakako Izumi Nakajima, Yoshihiko Hagiwara, Takahiro Oike, Ryuichi Okayasu, Takeshi Murakami, Takashi Nakano, Atsushi Shibata

PLOS ONE （PUBLIC LIBRARY SCIENCE） 10 （ 3 ） e0122582 2015年03月

研究論文（学術雑誌）, 共著, 最終著者, 責任著者, 査読有り, ISSN 1932-6203

The choice of DNA double strand break (DSB) repair pathway is determined at the stage of DSB end resection. Resection was proposed to control the balance between the two major DSB repair pathways, homologous recombination (HR) and non-homologous end joining (NHEJ). Here, we examined the regulation of DSB repair pathway choice at two-ended DSBs following ionizing radiation (IR) in G2 phase of the cell cycle. We found that cells pre-exposed to low-dose IR preferred to undergo HR following challenge IR in G2, whereas NHEJ repair kinetics in G1 were not affected by pre-IR treatment. Consistent with the increase in HR usage, the challenge IR induced Replication protein A (RPA) foci formation and RPA phosphorylation, a marker of resection, were enhanced by pre-IR. However, neither major DNA damage signals nor the status of core NHEJ proteins, which influence the choice of repair pathway, was significantly altered in pre-IR treated cells. Moreover, the increase in usage of HR due to pre-IR exposure was prevented by treatment with ATM inhibitor during the incubation period between pre-IR and challenge IR. Taken together, the results of our study suggest that the ATM-dependent damage response after pre-IR changes the cellular environment, possibly by regulating gene expression or post-transcriptional modifications in a manner that promotes resection.

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Carbon-Ion Beam Irradiation Kills X-Ray-Resistant p53-Null Cancer Cells by Inducing Mitotic Catastrophe

Napapat Amornwichet, Takahiro Oike, Atsushi Shibata, Hideaki Ogiwara, Naoto Tsuchiya, Motohiro Yamauchi, Yuka Saitoh, Ryota Sekine, Mayu Isono, Yukari Yoshida, Tatsuya Ohno, Takashi Kohno, Takashi Nakano

PLoS One （PUBLIC LIBRARY SCIENCE） 9 （ 12 ） e115121 2014年12月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 1932-6203

Background and Purpose: To understand the mechanisms involved in the strong killing effect of carbon-ion beam irradiation on cancer cells with TP53 tumor suppressor gene deficiencies.
Materials and Methods: DNA damage responses after carbon-ion beam or X-ray irradiation in isogenic HCT116 colorectal cancer cell lines with and without TP53 (p53(+/+) and p53(-/-), respectively) were analyzed as follows: cell survival by clonogenic assay, cell death modes by morphologic observation of DAPI-stained nuclei, DNA double-strand breaks (DSBs) by immunostaining of phosphorylated H2AX (gamma H2AX), and cell cycle by flow cytometry and immunostaining of Ser10-phosphorylated histone H3.
Results: The p53(-/-) cells were more resistant than the p53(+/+) cells to X-ray irradiation, while the sensitivities of the p53(+/+) and p53(-/-) cells to carbon-ion beam irradiation were comparable. X-ray and carbon-ion beam irradiations predominantly induced apoptosis of the p53(+/+) cells but not the p53(-/-) cells. In the p53(-/-) cells, carbon-ion beam irradiation, but not X-ray irradiation, markedly induced mitotic catastrophe that was associated with premature mitotic entry with harboring long-retained DSBs at 24 h post-irradiation.
Conclusions: Efficient induction of mitotic catastrophe in apoptosis-resistant p53-deficient cells implies a strong cancer cell-killing effect of carbon-ion beam irradiation that is independent of the p53 status, suggesting its biological advantage over X-ray treatment.

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DNA Double-Strand Break Repair Pathway Choice Is Directed by Distinct MRE11 Nuclease Activities

Atsushi Shibata, Davide Moiani, Andrew S. Arvai, Jefferson Perry, Shane M. Harding, Marie-Michelle Genois, Ranjan Maity, Sari van Rossum-Fikkert, Aryandi Kertokalio, Filippo Romoli, Amani Ismail, Ermal Ismalaj, Elena Petricci, Matthew J. Neale, Robert G. Bristow, Jean-Yves Masson, Claire Wyman, Penny A. Jeggo, John A. Tainer

MOLECULAR CELL （CELL PRESS） 53 （ 1 ） 7 - 18 2014年01月

研究論文（学術雑誌）, 共著, 筆頭著者, 査読有り, ISSN 1097-2765

MRE11 within the MRE11-RAD50-NBS1 (MRN) complex acts in DNA double-strand break repair (DSBR), detection, and signaling; yet, how its endo- and exonuclease activities regulate DSBR by non-homologous end-joining (NHEJ) versus homologous recombination (HR) remains enigmatic. Here, we employed structure-based design with a focused chemical library to discover specific MRE11 endo- or exonuclease inhibitors. With these inhibitors, we examined repair pathway choice at DSBs generated in G2 following radiation exposure. While nuclease inhibition impairs radiation-induced replication protein A (RPA) chromatin binding, suggesting diminished resection, the inhibitors surprisingly direct different repair outcomes. Endonuclease inhibition promotes NHEJ in lieu of HR, while exonuclease inhibition confers a repair defect. Collectively, the results describe nuclease-specific MRE11 inhibitors, define distinct nuclease roles in DSB repair, and support a mechanism whereby MRE11 endonuclease initiates resection, thereby licensing HR followed by MRE11 exonuclease and EXO1/BLM bidirectional resection toward and away from the DNA end, which commits to HR.

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Co-operation of BRCA1 and POH1 relieves the barriers posed by 53BP1 and RAP80 to resection

Andreas Kakarougkas, Amani Ismail, Yoko Katsuki, Raimundo Freire, Atsushi Shibata, Penny A. Jeggo

NUCLEIC ACIDS RESEARCH （OXFORD UNIV PRESS） 41 （ 22 ） 10298 - 10311 2013年09月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0305-1048

In G2 phase cells, DNA double-strand break repair switches from DNA non-homologous end-joining to homologous recombination. This switch demands the promotion of resection. We examine the changes in 53BP1 and RAP80 ionizing radiation induced foci (IRIF) in G2 phase, as these are factors that restrict resection. We observed a 2-fold increase in the volume of 53BP1 foci by 8 h, which is not seen in G1 cells. Additionally, an IRIF core devoid of 53BP1 arises where RPA foci form, with BRCA1 IRIF forming between 53BP1 and replication protein A (RPA). Ubiquitin chains assessed using alpha-FK2 antibodies are similarly repositioned. Repositioning of all these components requires BRCA1's BRCT but not the ring finger domain. 53BP1, RAP80 and ubiquitin chains are enlarged following POH1 depletion by small interfering RNA, but a devoid core does not form and RPA foci formation is impaired. Co-depletion of POH1 and RAP80, BRCC36 or ABRAXAS allows establishment of the 53BP1 and ubiquitin chain-devoid core. Thus, the barriers posed by 53BP1 and RAP80 are relieved by BRCA1 and POH1, respectively. Analysis of combined depletions shows that these represent distinct but interfacing barriers to promote loss of ubiquitin chains in the IRIF core, which is required for subsequent resection. We propose a model whereby BRCA1 impacts on 53BP1 to allow access of POH1 to RAP80. POH1-dependent removal of RAP80 within the IRIF core enables degradation of ubiquitin chains, which promotes loss of 53BP1. Thus, POH1 represents a novel component regulating the switch from non-homologous end-joining to homologous recombination.

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Opposing roles for 53BP1 during homologous recombination

Andreas Kakarougkas, Amani Ismail, Karolin Klement, Aaron A. Goodarzi, Sandro Conrad, Raimundo Freire, Atsushi Shibata, Markus Lobrich, Penny A. Jeggo

NUCLEIC ACIDS RESEARCH （OXFORD UNIV PRESS） 41 （ 21 ） 9719 - 9731 2013年08月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0305-1048

Although DNA non-homologous end-joining repairs most DNA double-strand breaks (DSBs) in G2 phase, late repairing DSBs undergo resection and repair by homologous recombination (HR). Based on parallels to the situation in G1 cells, previous work has suggested that DSBs that undergo repair by HR predominantly localize to regions of heterochromatin (HC). By using H3K9me3 and H4K20me3 to identify HC regions, we substantiate and extend previous evidence, suggesting that HC-DSBs undergo repair by HR. Next, we examine roles for 53BP1 and BRCA1 in this process. Previous studies have shown that 53BP1 is pro-non-homologous end-joining and anti-HR. Surprisingly, we demonstrate that in G2 phase, 53BP1 is required for HR at HC-DSBs with its role being to promote phosphorylated KAP-1 foci formation. BRCA1, in contrast, is dispensable for pKAP-1 foci formation but relieves the barrier caused by 53BP1. As 53BP1 is retained at irradiation-induced foci during HR, we propose that BRCA1 promotes displacement but retention of 53BP1 to allow resection and any necessary HC modifications to complete HR. In contrast to this role for 53BP1 in HR in G2 phase, we show that it is dispensable for HR in S phase, where HC regions are likely relaxed during replication.

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Visualisation of gamma H2AX Foci Caused by Heavy Ion Particle Traversal; Distinction between Core Track versus Non-Track Damage

Nakako Izumi Nakajima, Holly Brunton, Ritsuko Watanabe, Amruta Shrikhande, Ryoichi Hirayama, Naruhiro Matsufuji, Akira Fujimori, Takeshi Murakami, Ryuichi Okayasu, Penny Jeggo, Atsushi Shibata

PLOS ONE （PUBLIC LIBRARY SCIENCE） 8 （ 14 ） e70107 2013年08月

研究論文（学術雑誌）, 共著, 最終著者, 責任著者, 査読有り, ISSN 1932-6203

Heavy particle irradiation produces complex DNA double strand breaks (DSBs) which can arise from primary ionisation events within the particle trajectory. Additionally, secondary electrons, termed delta-electrons, which have a range of distributions can create low linear energy transfer (LET) damage within but also distant from the track. DNA damage by delta-electrons distant from the track has not previously been carefully characterised. Using imaging with deconvolution, we show that at 8 hours after exposure to Fe (similar to 200 keV/mu m) ions, gamma H2AX foci forming at DSBs within the particle track are large and encompass multiple smaller and closely localised foci, which we designate as clustered gamma H2AX foci. These foci are repaired with slow kinetics by DNA non-homologous end-joining (NHEJ) in G1 phase with the magnitude of complexity diminishing with time. These clustered foci (containing 10 or more individual foci) represent a signature of DSBs caused by high LET heavy particle radiation. We also identified simple gamma H2AX foci distant from the track, which resemble those arising after X-ray exposure, which we attribute to low LET delta-electron induced DSBs. They are rapidly repaired by NHEJ. Clustered gamma H2AX foci induced by heavy particle radiation cause prolonged checkpoint arrest compared to simple gamma H2AX foci following X-irradiation. However, mitotic entry was observed when similar to 10 clustered foci remain. Thus, cells can progress into mitosis with multiple clusters of DSBs following the traversal of a heavy particle.

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Endogenously induced DNA double strand breaks arise in heterochromatic DNA regions and require ataxia telangiectasia mutated and Artemis for their repair

Lisa Woodbine, H. Brunton, A. A. Goodarzi, A. Shibata, P. A. Jeggo

NUCLEIC ACIDS RESEARCH （OXFORD UNIV PRESS） 39 （ 16 ） 6986 - 6997 2011年09月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0305-1048

Ataxia telangiectasia (ATM) mutated and Artemis, the proteins defective in ataxia telangiectasia and a class of Radiosensitive-Severe Combined Immunodeficiency (RS-SCID), respectively, function in the repair of DNA double strand breaks (DSBs), which arise in heterochromatic DNA (HC-DSBs) following exposure to ionizing radiation (IR). Here, we examine whether they have protective roles against oxidative damage induced and/or endogenously induced DSBs. We show that DSBs generated following acute exposure of G0/G1 cells to the oxidative damaging agent, tert-butyl hydroperoxide (TBH), are repaired with fast and slow components of similar magnitude to IR-induced DSBs and have a similar requirement for ATM and Artemis. Strikingly, DSBs accumulate in ATM(-/-) mouse embryo fibroblasts (MEFs) and in ATM or Artemis-defective human primary fibroblasts maintained for prolonged periods under confluence arrest. The accumulated DSBs localize to HC-DNA regions. Collectively, the results provide strong evidence that oxidatively induced DSBs arise in HC as well as euchromatic DNA and that Artemis and ATM function in their repair. Additionally, we show that Artemis functions downstream of ATM and is dispensable for HC-relaxation and for pKAP-1 foci formation. These findings are important for evaluating the impact of endogenously arising DNA DSBs in ATM and Artemis-deficient patients.

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Analysis of Human Syndromes with Disordered Chromatin Reveals the Impact of Heterochromatin on the Efficacy of ATM-Dependent G(2)/M Checkpoint Arrest

Holly Brunton, Aaron A. Goodarzi, Angela T. Noon, Amruta Shrikhande, R. Scott Hansen, Penny A. Jeggo, Atsushi Shibata

MOLECULAR AND CELLULAR BIOLOGY （Taylor and Francis Group） 31 （ 19 ） 4022 - 4035 2011年07月

研究論文（学術雑誌）, 共著, 最終著者, 責任著者, 査読有り, ISSN 0270-7306

Heterochromatin (HC) poses a barrier to gamma H2AX focus expansion and DNA double-strand break (DSB) repair, the latter being relieved by ATM-dependent KAP-1 phosphorylation. Using high-resolution imaging, we show here that the HC superstructure markedly restricts ATM signaling to cell cycle checkpoint proteins. The impact of HC is greater than anticipated from the percentage of HC-DNA and, in distinction to DSB repair, ATM only partly overcomes the constraints posed by HC. Importantly, we examine ATM signaling in human syndromes with disordered HC. After depletion of MeCP2 and DNMT3B, proteins defective in the Rett and immunodeficiency with centromere instability and facial anomalies (ICF) syndromes, respectively, we demonstrate enhanced gamma H2AX signal expansion at HC-chromocenters in mouse NIH 3T3 cells, which have visible HC-chromocenters. Previous studies have shown that the G(2)/M checkpoint is inefficient requiring multiple DSBs to initiate arrest. MeCP2 and DNMT3B depletion leads to hypersensitive radiation-induced G(2)/M checkpoint arrest despite normal DSB repair. Cell lines from Rett, ICF, and Hutchinson-Guildford progeria syndrome patients similarly showed hyperactivated ATM signaling and hypersensitive and prolonged G(2)/M checkpoint arrest. Collectively, these findings reveal that heterochromatin contributes to the previously described inefficient G(2)/M checkpoint arrest and demonstrate how the signaling response can be uncoupled from DSB repair.

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Factors determining DNA double-strand break repair pathway choice in G2 phase

Atsushi Shibata, Sandro Conrad, Julie Birraux, Verena Geuting, Olivia Barton, Amani Ismail, Andreas Kakarougkas, Katheryn Meek, Gisela Taucher-Scholz, Markus Lobrich, Penny A. Jeggo

EMBO JOURNAL （EMBO press） 30 （ 6 ） 1079 - 1092 2011年05月

研究論文（学術雑誌）, 共著, 筆頭著者, 査読有り, ISSN 0261-4189

DNA non-homologous end joining (NHEJ) and homologous recombination (HR) function to repair DNA double-strand breaks (DSBs) in G2 phase with HR preferentially repairing heterochromatin-associated DSBs (HC-DSBs). Here, we examine the regulation of repair pathway usage at two-ended DSBs in G2. We identify the speed of DSB repair as a major component influencing repair pathway usage showing that DNA damage and chromatin complexity are factors influencing DSB repair rate and pathway choice. Loss of NHEJ proteins also slows DSB repair allowing increased resection. However, expression of an autophosphorylation-defective DNA-PKcs mutant, which binds DSBs but precludes the completion of NHEJ, dramatically reduces DSB end resection at all DSBs. In contrast, loss of HR does not impair repair by NHEJ although CtIP-dependent end resection precludes NHEJ usage. We propose that NHEJ initially attempts to repair DSBs and, if rapid rejoining does not ensue, then resection occurs promoting repair by HR. Finally, we identify novel roles for ATM in regulating DSB end resection; an indirect role in promoting KAP-1-dependent chromatin relaxation and a direct role in phosphorylating and activating CtIP. The EMBO Journal (2011) 30, 1079-1092. doi:10.1038/emboj.2011.27; Published online 11 February 2011

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Genomic DNA damage and ATR-Chk1 signaling determine oncolytic adenoviral efficacy in human ovarian cancer cells

Claire M. Connell, Atsushi Shibata, Laura A. Tookman, Kyra M. Archibald, Magdalena B. Flak, Katrina J. Pirlo, Michelle Lockley, Sally P. Wheatley, Iain A. McNeish

JOURNAL OF CLINICAL INVESTIGATION （American Society for Clinical Investigation） 121 （ 4 ） 1283 - 1297 2011年04月

研究論文（学術雑誌）, 共著, 筆頭著者, 査読有り, ISSN 0021-9738

Oncolytic adenoviruses replicate selectively within and lyse malignant cells. As such, they are being developed as anticancer therapeutics. However, the sensitivity of ovarian cancers to adenovirus cytotoxicity varies greatly, even in cells of similar infectivity. Using both the adenovirus E1A-CR2 deletion mutant dl922-947 and WT adenovirus serotype 5 in a panel of human ovarian cancer cell lines that cover a 3-log range of sensitivity, we observed profound overreplication of genomic DNA only in highly sensitive cell lines. This was associated with the presence of extensive genomic DNA damage. Inhibition of ataxia telangiectasia and Rad3-related checkpoint kinase 1 (ATR-Chk1), but not ataxia telangiectasia mutated (ATM), promoted genomic DNA damage and overreplication in resistant and partially sensitive cells. This was accompanied by increased adenovirus cytotoxicity both in vitro and in vivo in tumor-bearing mice. We also demonstrated that Cdc25A was upregulated in highly sensitive ovarian cancer cell lines after adenovirus infection and was stabilized after loss of Chk1 activity. Knockdown of Cdc25A inhibited virus-induced DNA damage in highly sensitive cells and blocked the effects of Chk1 inhibition in resistant cells. Finally, inhibition of Chk1 decreased homologous recombination repair of virus-induced genomic DNA double-strand breaks. Thus, virus-induced host cell DNA damage signaling and repair are key determinants of oncolytic adenoviral activity, and promoting unscheduled DNA synthesis and/or impeding homologous recombination repair could potentiate the effects of oncolytic adenoviruses in the treatment of ovarian cancer.

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