Role of p53 in radiation induced genomic instability

p53 在辐射诱导的基因组不稳定中的作用

• 批准号: 17201014
• 负责人: NIWA Ohtsura
• 金额: $ 27.04万
• 依托单位: National Institute of Radiological Sciences (2007)Kyoto University (2005-2006)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17201014/
• 关键词: p53; mouse pink-eyed unstable allele; homologous recombination; untargeted recombination; delayed recombination; Schizosaccharomvces pombe; DNA damage memory; p53; Sチェックポイント; 複製フォーク抑制; 相同組換え; 姉妹染色分体交換; ゲノム不安定性; 経世代影響; DNA複製フォーク進行; PCNA; プロ

We have previouslyreported the induction of mutations at the maternal alleles ofESTR locus and the pink-eyed unstable locus of Fl mice born to irradiated spermatozoa (PNAS 98, 1705, 2001; Radiat. Res. 157, 661, 2002)). These observations indicated the presence of cross-talk between X-irradiated paternal genome andunirradiated maternal genome whichinduces untargeted recombination in zygotic stage embryos and delayed recombination in fetuses. Further study has indicated that thiscross-talk is dependent on the function of p53 which mediates a novel p53 dependent S checkpoint in the zygotic stage mouse embryos (MCB, 22, 2220, 2002).In this study, we have first analyzed the p53 protein domain required for the novel p53 dependent S checkpoint Microinjection analyses of p53 protein with specific mutation demonstrated that this S checkpoint neither required transcription activation domain nor the ATM phosphorylation sites. However, the protein had mutation in the DNA binding domain was unable … More to carry out the S checkpoint function. These suggest that the p53 protein itself may bind to DNA to execute the S checkpoint (Oncogene24, 3229, 2005). In parallel with these results, DNA fiber analyses revealed that p53 dependent S checkpoint functioned in suppressing the progression of replication fork when cells were challenged by X-irradiationOOncogene25, 529, 2006).It can be envisaged that the slowing down of the replication fork progression may facilitate recombination between sisterchromatids. In order to test this possibility, primary mouse embryofibroblasts (MEFs) with the wild type p53 gene and those with the null allele were exposed to X-rays of up to 6 Gy. The frequency of SCE increased dose dependently in the wild type MEFs while that of the p53 null MEFs did not This links the p53 dependent S checkpoint to homologous recombination at least between sister chromatids.Our previous study indicated the increase in the frequency of recombinational mutation at the maternal pink-eyed unstable allele in the retinal pigment epithelial cells of sperm irradiated embryos. Since the retinal pigment epithelial cells start development at day 11 to day 12 of gestation, the frequency of recombination stays elevated for at least 11 to 12 days after fertilization with irradiated sperm. Indeed, the frequency of SCE was higher in primary MEFs of day 12 fetus fertilized by irradiated sperm (in preparation. Altogether, our 3 year mouse study has excavated a novel p53 dependent S checkpoint and its function toupregulate homologous recombination for at least 11 to 12 days in sperm irradiated embryos. In addition, they also demonstrated the DNA damage memory and its function in delayed recombination.In order to study the molecular mechanism, we have also tested Schizosaccharomyces pombe to see if the delayed and untargeted recombination can be induced. S. pombe exhibited upregulated recombination for around 10 cell cycle generations afterX-irradiation. The length of upregulated recombination was cell generation dependent rather than the absolute time dependent as shown by the temperature shift experiments. In addition, this upregulated recombination was not due to bystander factors since the phenomena were not affected by the presence of radical scavengers in the culture media. Concomitantly with the upregulation of the recombination frequency, Rad22 foci were observed for around 10 generations after irradiation. These studies also demonstrated the DNA damage memory and delayed recombination in S. pombe Less

我们以前曾报道过辐射精子所生F1小鼠的母系等位基因ESTR和粉眼不稳定基因的突变(PNAS 98,1705,2001；Radiat)。决议157,661,2002))。这些观察表明，X-射线照射的父本基因组和未辐照的母本基因组之间存在串扰，导致合子期胚胎的非靶向性重组和胎儿的延迟重组。进一步的研究表明，这种串扰依赖于P53的功能，P53介导了一个新的依赖于P53的S检查点。在本研究中，我们首先分析了新的依赖于P53的S检查点所需的P53蛋白结构域。对具有特定突变的P53蛋白的显微注射分析表明，这个S检查点既不需要转录激活结构域，也不需要ATM磷酸化位点。然而，该蛋白在dna结合区发生了突变，无法…更多的是要履行S关卡的职能。这些结果表明，P53蛋白本身可能与脱氧核糖核酸结合来执行S检查点(Oncogen24,3229,2005)。与这些结果相平行的是，DNA纤维分析显示，当细胞受到X-射线的挑战时，依赖于p53的S检查点在抑制复制分叉的进展中发挥作用(见Oncogen25,529,2006)。可以预见，复制分叉进展的减慢可能促进姐妹染色单体之间的重组。为了测试这种可能性，携带野生型p53基因的原代小鼠胚胎成纤维细胞(MEF)和那些零等位基因的小鼠胚胎成纤维细胞(MEF)被暴露在高达6GyX射线下。野生型MEF的姐妹染色单体交换频率呈剂量依赖性增加，而p53缺失型MEF的姐妹染色单体姐妹染色单体间的同源重组与依赖于P53的S检查点无关。我们先前的研究表明，精子照射胚胎视网膜色素上皮细胞中红眼不稳定等位基因的重组突变频率增加。由于视网膜色素上皮细胞在妊娠第11天至第12天开始发育，重组的频率在与辐射精子受精后至少11至12天内保持较高水平。事实上，经辐照精子受精的第12天胎儿的原始MEF的姐妹染色单体交换频率更高(在准备阶段)。总之，我们3年的小鼠研究发现了一个新的依赖于P53的S检查点，它的功能在精子照射的胚胎中至少进行了11-12天的同源重组。此外，他们还展示了DNA损伤记忆及其在延迟重组中的作用。为了研究其分子机制，我们还对裂殖酵母进行了检测，看看是否可以诱导延迟的和非靶向的重组。经X-射线照射后，约10代细胞周期中，S.pombe表现出上调重组。温移实验表明，上调重组的长度依赖于细胞世代，而不是依赖于绝对时间。此外，这种上调的重组不是由于旁观者因素，因为这种现象不受培养基中存在的自由基清除剂的影响。伴随着重组频率的上调，照射后观察到了10代左右的Rad22病灶。这些研究也证明了金黄色葡萄球菌DNA损伤记忆和延迟重组

项目成果

Radiation induction of delayed recombination in S. pombe.

粟酒裂殖酵母延迟重组的辐射诱导。

• 期刊: DNA Repair (in press)
• 作者: Takada J;uematsu N;Shiraishi S;Toyoshima M;Matsumoto T;Niwa O.
• 通讯作者: Niwa O.

Ku70/80 Modulates ATM and ATR Signaling Pathway in Response DNA Double Strand Breaks. [Poster presentation and also selected for workshop presentation

Ku70/80 在响应 DNA 双链断裂中调节 ATM 和 ATR 信号通路。

• 发表时间: 2007
• 作者: Tokoro T.;Watanabe A.;Kayanne H.;Nadaoka K.;Tamura H.;Nozaki K.;Kato K.;Negishi A.;丹羽 太貫;Igarashi,Y.;丹羽太貫;室崎 将史;丹羽太貫;Sato,E.;丹羽 太貫;Kuji,M.;栗政 明弘;Watanabe,K.;栗政 明弘
• 通讯作者: 栗政 明弘

Suppression of replication fork progression in low dose specific p53 dependent S-phase DNA damage checkpoint.

低剂量特异性 p53 依赖性 S 期 DNA 损伤检查点复制叉进展的抑制。

• 发表时间: 2006
• 期刊: Oncogene 25(44)
• 作者: Shimura T;Toyoshima M;Adiga SK;Kunoh T;Nagai H;Shimizu N;Inoue M;Niwa O.
• 通讯作者: Niwa O.

Early molecular events in preimplantation stage mouse embryos born to irradiated sperm

受辐射精子所生的植入前阶段小鼠胚胎的早期分子事件

• 发表时间: 2007
• 作者: Tokoro T.;Watanabe A.;Kayanne H.;Nadaoka K.;Tamura H.;Nozaki K.;Kato K.;Negishi A.;丹羽 太貫;Igarashi,Y.;丹羽太貫;室崎 将史;丹羽太貫
• 通讯作者: 丹羽太貫

Characterization of novel murine anti-CD20 monoclonal antibodies and their comparison to 2B8 and c2B8 (rituximab).

• DOI: 10.3892/ijo.31.1.29
• 发表时间: 2007-07
• 期刊: International journal of oncology
• 影响因子: 5.2
• 作者: Michio Nishida;S. Usuda;M. Okabe;H. Miyakoda;M. Komatsu;Hiroshi Hanaoka;K. Teshigawara;O. Niwa