p53 dependent S checkpoint in early mouse embryos and radiation induction of genomic instability

• Project/Area Number: 14208067
• Research Category: Grant-in-Aid for Scientific Research (A)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: 環境影響評価(含放射線生物学)
• Research Institution: Kyoto University
• Principal Investigator: NIWA Ohtsura Kyoto University, Radiation Biology Center, Professor, 放射線生物研究センター, 教授 (80093293)
• Project Period (FY): 2002 – 2004
• Project Status: Completed (Fiscal Year 2004)
• Budget Amount: ¥21,450,000 (Direct Cost: ¥16,500,000、Indirect Cost: ¥4,950,000); Fiscal Year 2004: ¥6,240,000 (Direct Cost: ¥4,800,000、Indirect Cost: ¥1,440,000); Fiscal Year 2003: ¥6,240,000 (Direct Cost: ¥4,800,000、Indirect Cost: ¥1,440,000); Fiscal Year 2002: ¥8,970,000 (Direct Cost: ¥6,900,000、Indirect Cost: ¥2,070,000)
• Keywords: p53 dependent S checkpoint / suppression of replication fork progression / transactivation independent / DNA binding domain / ATM dependency / sperm irradiated zygotes / pink eyed unstable allele / reversion mutation / ゲノム防御機構 / マウス初期胚 / 遅延アポトーシス / 遅延細胞周期抑制 / p21 / ATM / PCNA / p53 / NBS / Sチェックポイント / PCNAフォーカス

Research Abstract

Elevated levels of mutations were found at the maternally derived alleles of the Ms6hm minisatellite locus and the pink-eyed unstable locus in F1 mice born to male mice irradiated at the spermatozoa stage. These mutations demonstrated the operation of untargeted and delayed recombinational mutations due to genomic instability which was induced during early mouse development by radiation DNA damage introduced by the irradiated sperm. In order to elucidate the molecular mechanism of genomic instability, we have undertaken studies of damage response in sperm irradiated mouse embryos.
A p53 responsible reporter plasmid microinjected into zygotes was transcriptionally activated when they zygotes were fertilized with irradiated sperm. This pronuclear cross-talk was found to suppress DNA synthesis of female pronucleus as well as of male pronucleus in sperm irradiated zygotes. This suppression of DNA synthesis was not observed in p53-/-zygotes which was restored when the sperm irradiated p53-/-zygotes were microinjected with p53 protein, suggesting a novel pathway of S checkpoints. Microinjection of mutant p53 proteins into sperm irradiated p53-/-zygotes demonstrated that this p53 dependent S checkpoint required DNA binding domain of p53,but the transactivation domain.
The p53 dependent S checkpoint was analyzed in primary mouse and found operate at doses below 2 Gy. In addition, analyses of DNA fiber elongation by double labeling the cells with Id U and Cld U showedthatthe p53 dependent S checkpoint suppressed DNA synthesis by slowing down of replication fork progression. This slowing down of replication was associated with a higher rate of recombination between sister chromatids which can explain the untargeted recombination of minisatellite. Delayed recombination observed at the pink-eyed unstable allele requires yet another mechanism of damage memory for which much more to be studied yet.

Research Products

• [Journal Article] Transcription independent suppression of DNA synthesis by p53 in sperm irradiated mouse zygotes. (2005)
  • Author(s): Toyoshima M et al.
  • Journal Title: Oncogene 24 Pages: 3229-3235
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Transcription independent suppression of DNA synthesis by p53 in sperm irradiated mouse zygotes. (2005)
  • Author(s): Toyoshima M, Shimura T, Adiga SK, Taga M, Shiraishi K, Inoue M, Yuan Z-M, Niwa O.
  • Journal Title: Oncogene 24 Pages: 3229-3235
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Transcription independent suppression of DNA synthesis by p53 in sperm irradiated mouse zygotes. (2005)
  • Author(s): Toyoshima M et al.
  • Journal Title: Oncogene (印刷中)
• [Journal Article] Comparison of properies of spontaneous and radiation induced mouse thymic lymphomas : Role of Trp53 and radiation. (2005)
  • Author(s): Kubota T et al.
  • Journal Title: Radiation Research 163 Pages: 159-164
• [Journal Article] Atm-heterozygous deficiency enhances development of mammary carcinomas in p53-heterozygous knockout mice. (2005)
  • Author(s): Umesato S et al.
  • Journal Title: Breast Cancer Research 7 Pages: 164-170
• [Journal Article] Predisposition to mouse thymic lymphomas in response to ionizing radiation depends on variant alleles encoding metal-responsive transcription factor-1 (Mtf-1). (2005)
  • Author(s): Tamura Y et al.
  • Journal Title: Oncogene 24 Pages: 399-406
• [Journal Article] Generation of multipotent stem cells from postnatal mouse testis (2004)
  • Author(s): Kanatsu-Shinohara M et al.
  • Journal Title: Cell 119 Pages: 1001-1012
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Predisposition to mouse thymic lymphomas in response to ionizing radiation depends on variant alleles encoding metal-responsive transcription factor-1 (Mtf-1) (2004)
  • Author(s): Tamura Y et al.
  • Journal Title: Oncogene 24 Pages: 399-406
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Generation of multipotent stem cells from postnatal mouse testis. (2004)
  • Author(s): Kanatsu-Shinohara M, Inoue K, Lee J, Yoshimoto M, Ogonuki N, Miki H, Kato T, Baba S, Kazuki Y, Toyokuni S, Toyoshima M, Niwa O, Oshimura M, Heike T, Ishino F, Nakahata T, Ogura A, Shinohara, T.
  • Journal Title: Cell 119 Pages: 1001-1012
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Predisposition to mouse thymic lymphomas in response to ionizing radiation depends on variant alleles encoding metal-responsive transcription factor-1(Mtf-1). (2004)
  • Author(s): Tamura Y, Maruyama M, Mishima Y, Fujisawa H, Obata M, Kodama Y, Yoshikai Y, Aoyagi Y, Niwa O, Schaffner W, Kominami R.
  • Journal Title: Oncogene 24 Pages: 399-406
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] RERF : the monument and the crisis (2004)
  • Author(s): Niwa O
  • Journal Title: Radiat.Res. 162 Pages: 105-105
• [Journal Article] Induced genomic instability in irradiated germ cells and in the offspring ; reconciling discrepancies among the human and animal studies. (2003)
  • Author(s): Niwa, O
  • Journal Title: Oncogene 22 Pages: 7078-7086
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Bcl11b is required for differentiation and survival of ab T lymphocytes (2003)
  • Author(s): Wakabayashi, Y et al.
  • Journal Title: Nature Immunol. 4 Pages: 533-539
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Induced genomic instability in irradiated germ cells and in the offspring ; reconciling discrepancies among the human and animal studies. (2003)
  • Author(s): Niwa, O.
  • Journal Title: Oncogene 22 Pages: 7078-7086
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Bcll1b is required for differentiation and survival of ab T lymphocytes. (2003)
  • Author(s): Wakabayashi, Y., J.Inoue, J.Sakata, Y.Takahashi, A.Matsuki, H.Kosugi-Okano, Y.Miyazawa, Y.Saito, H.Watanabe, Y.Mixhims, O.Niwa, R.Kominami
  • Journal Title: Nature Immunol. 4 Pages: 533-539
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] p53 dependent S phase damage checkpoint and pronuclear crosstalk in mouse zygotes with X-irradiated sperm (2002)
  • Author(s): Shimura, T et al.
  • Journal Title: Mol.Cell.Biol. 22 Pages: 2220-2228
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] p53 dependent S phase damage checkpoint and pronuclear crosstalk in mouse zygotes with X-irradiated sperm. (2002)
  • Author(s): Shimura, T., M.Inoue, M.Taga, K.Shiraishi, N.Uematsu, N.Takei, Z.Yuan, O.Niwa
  • Journal Title: Mol.Cell.Biol. 22 Pages: 2220-2228
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Satish Kumar Adiga: "Delayed activation of p21 and its protective role in preimplantation stage mouse embryos fertilized with irradiated sperm."発表予定.
• [Publications] 丹羽太貫: "The D5Mit7 locus on mouse chromosome 5 provides resistance to g-ray induced, but not N-methyl-N-nitrosourea-induced thymic iymphomas."Carcinogenesis. 143-148 (2004)
• [Publications] 丹羽太貫: "Intragenic deletions of Rit1/Bcl11b tumor suppressor gene in g-ray-induced thymic lymphomas and normal thymus caused by V(D)J recombinase activity."Carcinogenesis. 25. 1-7 (2004)
• [Publications] 丹羽太貫: "Induced genomic instability in irradiated germ cells and in the offspring ; reconciling discrepancies among the human and animal studies."Oncogene. 22. 7078-7086 (2003)
• [Publications] 丹羽太貫: "Bcl11b is required for differentiation and survival of ab T lymphocytes."Nature Immunol.. 4. 533-539 (2003)
• [Publications] 丹羽太貫: "Mapping of genetic modifiers of thymic lymphoma development in p53-knockout mice. 333"Oncogene. 1098-1102 (2003)
• [Publications] 丹羽太貫: "Bcl11b is required for differentiation and survival of αΒ T lymphocytes"Nature Immunology. (in press).
• [Publications] 丹羽太貫: "Mapping of genetic modifiers of thymic lymphoma development in p53-knockout mice"Oncogene. 22. 1098-1102 (2003)
• [Publications] 丹羽太貫: "Homologous deletions and poit mutations of the Rit1/Ncl11b gene in γ-ray induced mouse thymic lymphomas"Biochem.Biophys.Res.Commu. 301. 598-603 (2003)
• [Publications] 丹羽太貫: "Novel surveilance of the 53-depenent S checkpoint ensures chromosome damage repair and development of mouse embryos fertilized with X-irradiated sperm"Radiat. Res.. 158. 735-742 (2002)
• [Publications] 丹羽太貫: "Persistent induction of somatic reversions of the pink-eyed unstable mutation in F1 mice born to fathers irradiated at the spermatozoa stage"Radiat. Res.. 157. 661-667 (2002)
• [Publications] 丹羽太貫: "p53 dependent S phase damage checkpoint and pronuclear crosstalk in mouse zygotes with X-irradiated sperm"Mol. Cell. Biol.. 22. 2220-2228 (2002)