Papers - Shibata, Atsushi

Role of ATM and the Damage Response Mediator Proteins 53BP1 and MDC1 in the Maintenance of G(2)/M Checkpoint Arrest

Atsushi Shibata, Olivia Barton, Angela T. Noon, Kirsten Dahm, Dorothee Deckbar, Aaron A. Goodarzi, Markus Loebrich, Penny A. Jeggo

MOLECULAR AND CELLULAR BIOLOGY (the American Society for Microbiology)  30 ( 13 ) 3371 - 3383 2010.07

Research paper (scientific journal), Joint Work, Lead author, Accepted,  ISSN  0270-7306

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ATM-dependent initiation of the radiation-induced G(2)/M checkpoint arrest is well established. Recent results have shown that the majority of DNA double-strand breaks (DSBs) in G(2) phase are repaired by DNA nonhomologous end joining (NHEJ), while similar to 15% of DSBs are slowly repaired by homologous recombination. Here, we evaluate how the G(2)/M checkpoint is maintained in irradiated G(2) cells, in light of our current understanding of G(2) phase DSB repair. We show that ATM-dependent resection at a subset of DSBs leads to ATR-dependent Chk1 activation. ATR-Seckel syndrome cells, which fail to efficiently activate Chk1, and small interfering RNA (siRNA) Chk1-treated cells show premature mitotic entry. Thus, Chk1 significantly contributes to maintaining checkpoint arrest. Second, sustained ATM signaling to Chk2 contributes, particularly when NHEJ is impaired by XLF deficiency. We also show that cells lacking the mediator proteins 53BP1 and MDC1 initially arrest following radiation doses greater than 3 Gy but are subsequently released prematurely. Thus, 53BP1(-/-) and MDC1(-/-) cells manifest a checkpoint defect at high doses. This failure to maintain arrest is due to diminished Chk1 activation and a decreased ability to sustain ATM-Chk2 signaling. The combined repair and checkpoint defects conferred by 53BP1 and MDC1 deficiency act synergistically to enhance chromosome breakage.

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Sensitization to Radiation and Alkylating Agents by Inhibitors of Poly(ADP-ribose) Polymerase Is Enhanced in Cells Deficient in DNA Double-Strand Break Repair

Dana A. Loeser, Atsushi Shibata, Akiko K. Shibata, Lisa J. Woodbine, Penny A. Jeggo, Anthony J. Chalmers

MOLECULAR CANCER THERAPEUTICS (American )  9 ( 6 ) 1775 - 1787 2010.06

Joint Work, Lead author, Accepted,  ISSN  1535-7163

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As single agents, chemical inhibitors of poly(ADP-ribose) polymerase (PARP) are nontoxic and have clinical efficacy against BRCA1- and BRCA2-deficient tumors. PARP inhibitors also enhance the cytotoxicity of ionizing radiation and alkylating agents but will only improve clinical outcomes if tumor sensitization exceeds effects on normal tissues. It is unclear how tumor DNA repair proficiency affects the degree of sensitization. We have previously shown that the radiosensitizing effect of PARP inhibition requires DNA replication and will therefore affect rapidly proliferating tumors more than normal tissues. Because many tumors exhibit defective DNA repair, we investigated the impact of double-strand break (DSB) repair integrity on the sensitizing effects of the PARP inhibitor olaparib. Sensitization to ionizing radiation and the alkylating agent methylmethane sulfonate was enhanced in DSB repair-deficient cells. In Artemis(-/-) and ATM(-/-) mouse embryo fibroblasts, sensitization was replication dependent and associated with defective repair of replication-associated damage. Radiosensitization of Ligase IV(-/-) mouse embryo fibroblasts was independent of DNA replication and is explained by inhibition of "alternative" end joining. After methylmethane sulfonate treatment, PARP inhibition promoted replication-independent accumulation of DSB, repair of which required Ligase IV. Our findings predict that the sensitizing effects of PARP inhibitors will be more pronounced in rapidly dividing and/or DNA repair defective tumors than normal tissues and show their potential to enhance the therapeutic ratio achieved by conventional DNA-damaging agents. Mol Cancer Ther; 9(6); 1775-87. (C)2010 AACR.

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53BP1-dependent robust localized KAP-1 phosphorylation is essential for heterochromatic DNA double-strand break repair

Angela T. Noon, Atsushi Shibata, Nicole Rief, Markus Loebrich, Grant S. Stewart, Penelope A. Jeggo, Aaron A. Goodarzi

NATURE CELL BIOLOGY (NATURE PUBLISHING GROUP)  12 ( 2 ) 177 - 184 2010.02

Research paper (scientific journal), Joint Work, Accepted,  ISSN  1465-7392

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DNA double-strand breaks (DSBs) trigger ATM (ataxia telangiectasia mutated) signalling and elicit genomic rearrangements and chromosomal fragmentation if misrepaired or unrepaired. Although most DSB repair is ATM-independent, similar to 15% of ionizing radiation (IR)-induced breaks persist in the absence of ATM-signalling(1). 53BP1 (p53-binding protein 1) facilitates ATM-dependent DSB repair but is largely dispensable for ATM activation or checkpoint arrest. ATM promotes DSB repair within heterochromatin by phosphorylating KAP-1 (KRAB-associated protein 1, also known as TIF1 beta, TRIM28 or KRIP-1; ref. 2). Here, we show that the ATM signalling mediator proteins MDC1, RNF8, RNF168 and 53BP1 are also required for heterochromatic DSB repair. Although KAP-1 phosphorylation is critical for 53BP1-mediated repair, overall phosphorylated KAP-1 (pKAP-1) levels are only modestly affected by 53BP1 loss. pKAP-1 is transiently pan-nuclear but also forms foci overlapping with gamma H2AX in heterochromatin. Cells that do not form 53BP1 foci, including human RIDDLE (radiosensitivity, immunodeficiency, dysmorphic features and learning difficulties) syndrome cells, fail to form pKAP-1 foci. 53BP1 amplifies Mre11-NBS1 accumulation at late-repairing DSBs, concentrating active ATM and leading to robust, localized pKAP-1. We propose that ionizing-radiation induced foci (IRIF) spatially concentrate ATM activity to promote localized alterations in regions of chromatin otherwise inhibitory to repair.

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ATM and Artemis promote homologous recombination of radiation-induced DNA double-strand breaks in G2

Andrea Beucher, Julie Birraux, Leopoldine Tchouandong, Olivia Barton, Atsushi Shibata, Sandro Conrad, Aaron A. Goodarzi, Andrea Krempler, Penny A. Jeggo, Markus Loebrich

EMBO JOURNAL (EMBO Press)  28   3413 - 3427 2009.11

Joint Work, Accepted,  ISSN  0261-4189

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Homologous recombination (HR) and non-homologous end joining (NHEJ) represent distinct pathways for repairing DNA double-strand breaks (DSBs). Previous work implicated Artemis and ATM in an NHEJ-dependent process, which repairs a defined subset of radiation-induced DSBs in G1-phase. Here, we show that in G2, as in G1, NHEJ represents the major DSB-repair pathway whereas HR is only essential for repair of similar to 15% of X-or gamma-ray-induced DSBs. In addition to requiring the known HR proteins, Brca2, Rad51 and Rad54, repair of radiation-induced DSBs by HR in G2 also involves Artemis and ATM suggesting that they promote NHEJ during G1 but HR during G2. The dependency for ATM for repair is relieved by depleting KAP-1, providing evidence that HR in G2 repairs heterochromatin-associated DSBs. Although not core HR proteins, ATM and Artemis are required for efficient formation of single-stranded DNA and Rad51 foci at radiation-induced DSBs in G2 with Artemis function requiring its endonuclease activity. We suggest that Artemis endonuclease removes lesions or secondary structures, which inhibit end resection and preclude the completion of HR or NHEJ. The EMBO Journal (2009) 28, 3413-3427. doi: 10.1038/emboj.2009.276; Published online 24 September 2009

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Role of Parp-1 in suppressing spontaneous deletion mutation in the liver and brain of mice at adolescence and advanced age

Atsushi Shibata, Daisuke Maeda, Hideki Ogino, Masahiro Tsutsumi, Takehiko Nohmi, Hitoshi Nakagama, Takashi Sugimura, Hirobumi Teraoka, Mitsuko Masutani

MUTATION RESEARCH-FUNDAMENTAL AND MOLECULAR MECHANISMS OF MUTAGENESIS (ELSEVIER SCIENCE BV)  664   20 - 27 2009.05

Research paper (scientific journal), Joint Work, Lead author, Accepted,  ISSN  0027-5107

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Poly(ADP-ribose) polymerase-1 knockout (Parp-1(-/-)) mice show increased frequency of spontaneous liver tumors compared to wild-type mice after aging. To understand the impact of Parp-1 deficiency on mutations during aging, in this study, we analyzed spontaneous mutations in Parp-1(-/-) aged mice. Parp-1(-/-) mice showed tendencies of higher mutation frequencies of the red/gam genes at 18 months of age, compared to Parp-1(+/+) mice, in the liver and brain. Complex-type deletions, accompanying small insertion were observed only in Parp-1(-/-) mice in the liver and brain. Further analysis in the liver showed that the frequency of single base deletion mutations at non-repeat or short repeat sequences was 5.8-fold higher in Parp-1(-/-) than in Parp-1(+/+) mice (p < 0.05). A 3.2-fold higher tendency of the deletion frequency of two bases or more was observed in Parp-1(-/-) mice compared to Parp-1(+/+) mice (p = 0.084). These results support the model that Parp-1 is involved in suppressing imprecise repair of endogenous DNA damage leading to deletion mutation during aging. The mutation frequencies of the gpt gene in the brain were found to be 3-fold lower in Parp-1(-/-) than in Parp-1(+/+) mice at 4 months of age (p < 0.01), implying that Parp-1 may be positively involved in imprecise DNA repair in the brain. On the other hand, the frequencies of gpt mutation showed an increase at 18 months of age in the Parp-1(-/-) (p < 0.05) but not in Parp-1 brains, suggesting that Parp-1 deficiency causes an increase of point mutations in the brain by aging. (c) 2009 Elsevier B.V. All rights reserved.

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The Maintenance of ATM Dependent G2/M Checkpoint Arrest Following Exposure to Ionizing Radiation

Atsushi Shibata, Olivia Barton, Angela T. Noon, Kirsten Dahm, Dorothee Deckbar, Aaron A. Goodarzi, Markus Lobrich, Penny A. Jeggo

Acta Medica Nagasakiensia (長崎大学医学部)   ( 53 ) 19 - 21 2009.03

Research paper (bulletin of university, research institution), Joint Work, Lead author, Accepted,  ISSN  00016055

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The G2/M checkpoint is important in preventing cells with unrepaired DNA double strand breaks (DSBs) entering mitosis, an event which is
likely to result in genomic instability. We recently reported that heckpoint arrest is maintained until close to completion of DSB repair and that the duration of checkpoint arrest depends on the dose and DSB repair capacity rather than lasting for a fixed period of time. ATM leads to phosphorylation of Chk1/2 inG2 phase following exposure to ionizing radiation. These transducer kinases can phosphorylate and inhibit Cdc25
activity, which is the phosphatase regulatingmitotic entry. In this study we dissect three processes that contribute to the maintenance of checkpoint arrest in irradiated G2 phase cells. First, the ATR-Chk1 pathway contributes to maintaining checkpoint arrest, although it is dispensable for the initial activation of checkpoint arrest. Second, ongoing ATM to Chk2 signalling from unrepaired DSBs contributes to checkpoint arrest. This process plays a greater role in a repair defective background. Finally, slow decay of the initially activated Chk2 also contributes to the maintenance of checkpoint arrest. 53BP1 and MDC1 defective cells show an initial checkpoint defect after low doses but are proficient in initial activation of arrest after high doses. After higher radiation doses, however, 53BP1– /– and MDC1–/– MEFs fail to maintain checkpoint arrest. Furthermore 53BP1– /– andMDC1–/ –MEFs display elevated mitotic breakage even after high doses.We show that the defect in the maintenance of checkpoint arrest conferred by 53BP1 and MDC1 deficiency substantially enhances chromosome breakage.

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Differential involvement of phosphatidylinositol 3-kinase-related protein kinases in hyperphosphorylation of replication protein A2 in response to replication-mediated DNA double-strand breaks.

Ryo Sakasai, Keitaro Shinohe, Yosuke Ichijima, Naoyuki Okita, Atsushi Shibata, Kinji Asahina, Hirobumi Teraoka

Genes to cells : devoted to molecular & cellular mechanisms (Wiley-Blackwell Publishing Ltd.)  11 ( 3 ) 237 - 246 2006.03

Research paper (scientific journal), Joint Work, Accepted,  ISSN  1356-9597

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Replication protein A2 (RPA2), a component of the RPA heterotrimer, is hyperphosphorylated and forms nuclear foci in response to camptothecin (CPT) that directly induces replication-mediated DNA double-strand breaks (DSBs). Ataxia-telangiectasia mutated and Rad3-related kinase (ATR) and DNA-dependent protein kinase (DNA-PK) are activated by CPT, and RPA2 is hyperphosphorylated in a DNA-PK-dependent manner. To distinguish the roles of phosphatidylinositol 3-kinase-related protein kinases including DNA-PK, ataxia-telangiectasia mutated (ATM), and ATR, in the response to replication-mediated DSBs, we analyzed RPA2 focus formation and hyperphosphorylation during exposure to CPT. ATR knock-down with siRNA suppressed CPT-induced RPA2 hyperphosphorylation and focus formation. CPT-induced RPA2 focus formation was normally observed in DNA-PK- or ATM-deficient cells. Comparison between CPT and hydroxyurea (HU) indirectly inducing DSBs showed that RPA2 hyperphosphorylation is DNA-PK-dependent in CPT-treated cells and DNA-PK-independent in HU-treated cells. Although RPA2 foci rapidly formed in response to HU and CPT, the RPA2 hyperphosphorylation in HU-treated cells occurred later than in the CPT-treated cells, indicating that the DNA-PK dependency of RPA2 hyperphosphorylation is likely to be related to the mode of DSB induction. These results suggest that DNA-PK is responsible for the RPA2 hyperphosphorylation following ATR-dependent RPA2 focus formation in response to replication-mediated DSBs directly induced by CPT.

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Parp-1 deficiency causes an increase of deletion mutations and insertions/rearrangements in vivo after treatment with an alkylating agent

A Shibata, N Kamada, K Masumura, T Nohmi, S Kobayashi, H Teraoka, H Nakagama, T Sugimura, H Suzuki, M Masutani

ONCOGENE (NATURE PUBLISHING GROUP)  24   1328 - 1337 2005.02

Research paper (scientific journal), Joint Work, Lead author, Accepted,  ISSN  0950-9232

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Accumulated evidence suggests that Parp-1 is involved in DNA repair processes, including base excision repair, single-strand and double-strand break repairs. To understand the precise role of Parp-1 in genomic stability in vivo, we carried out mutation analysis using Parp-1 knockout (Parp-1(-/-)) mice harboring two marker genes, gpt and red/gam genes. Spontaneous mutant frequencies of both genes in the bone marrows and livers did not differ significantly between Parp-1(-/-) and Parp-1(+/+) mice (P > 0.05). After treatment with an alkylating agent, N-nitrosobis(2-hydroxypropyl)amine (BHP), the mutant frequency of the red/gam genes in the liver in Parp-1(-/-) mice was 1.6-fold higher than that in Parp-1(+/+) mice (P<0.05). Categorization of the mutations revealed that deletions larger than 1 kb or those accompanying 1-5 lip insertions at the deletion junctions, as well as rearrangements, were more frequently observed in Parp-1(-/-) than in Parp-1(+/+) mice (P<0.05, respectively). In contrast, mutant frequencies of the gpt gene in the livers of Parp-1(-/-) and Parp-1(+/+) mice after BHP treatment were both elevated and there was no significant difference between the genotypes. These results indicate that Parp-I is implicated in suppressing deletion mutations in vivo, especially those accompanying small insertions or rearrangements. Published online 20 December 2004.

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Efficient method for mapping and characterizing structures of deletion mutations in gpt delta mice using Southern blot analysis with oligo DNA probes

A Shibata, M Masutani, N Kamada, K Masumura, H Nakagama, S Kobayashi, H Teraoka, H Suzuki, T Nohmi

ENVIRONMENTAL AND MOLECULAR MUTAGENESIS (WILEY-LISS)  43 ( 3 ) 204 - 207 2004.01

Research paper (scientific journal), Joint Work, Lead author, Accepted,  ISSN  0893-6692

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Improvement of the Spi(-) assay for mutations in gpt delta mice by including magnesium ions during plaque formation

A Shibata, M Masutani, T Nozaki, N Kamada, H Fujihara, K Masumura, H Nakagama, T Sugimura, S Kobayashi, H Suzuki, T Nohmi

ENVIRONMENTAL AND MOLECULAR MUTAGENESIS (WILEY-LISS)  41 ( 5 ) 370 - 372 2003.06

Research paper (scientific journal), Joint Work, Lead author, Accepted,  ISSN  0893-6692

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Inhibition of poly(ADP-ribose) glycohydrolase activity by cyclic peptide antibiotics containing piperazic acid residues

Mitsuko Masutani, Takashi Shimokawa, Masayuki Igarashi, Masa Hamada, Atsushi Shibata, Sachiho Oami, Tadashige Nozaki, Hitoshi Nakagama, Takashi Sugimura, Tomio Takeuchi, Makoto Hori

Proceedings of the Japan Academy Series B: Physical and Biological Sciences (日本学士院)  78 ( 1 ) 15 - 17 2002.01

Research paper (scientific journal), Joint Work, Accepted,  ISSN  0386-2208

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A member of the cyclic hexadepsipeptide family, PD124,966, isolated from a fermentation product of an actinomycete, inhibited poly(ADP-ribose) glycohydrolase (Parg) with IC50 of 40 μg/ml. A novel cyclic peptide, pargamicin, isolated from a fermentation broth of Amicolatopsis sp., also inhibited Parg with IC50 of 28 μg/ml. A linear peptide containing piperazic acid residues, piperastatin A, isolated from Streptomyces lavendofoliae, did not inhibit Parg activity.

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