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

基本信息

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

项目摘要

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

我们之前报道过受辐射精子所生的 F1 小鼠的 ESTR 基因座和红眼不稳定基因座的母体等位基因的突变诱导（PNAS 98, 1705, 2001；Radiat. Res. 157, 661, 2002））。表明X射线照射的父本基因组和未照射的母本基因组之间存在串扰，从而导致非靶向合子期胚胎中的重组和胎儿中的延迟重组进一步研究表明，这种串扰依赖于 p53 的功能，p53 在合子期小鼠胚胎中介导新的 p53 依赖性 S 检查点 (MCB, 22, 2220, 2002)。在本研究中，我们首先分析了 p53 蛋白的新型 p53 依赖性 S 检查点显微注射分析所需的 p53 蛋白结构域。特异性突变表明该 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，但是，该蛋白质的 DNA 结合结构域发生突变，无法执行 S 检查点功能。这表明 p53 蛋白本身可能结合。与这些结果平行，DNA 纤维分析表明，当细胞受到攻击时，p53 依赖性 S 检查点可以抑制复制叉的进展。通过 X-irradiationOOncogene25, 529, 2006)。可以设想，复制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，具有野生型 p53 基因的原代小鼠胚胎成纤维细胞 (MEF) 和具有无效等位基因的个体暴露于高达 6 Gy 的 X 射线。野生型 MEF 中 SCE 的频率呈剂量依赖性增加。这将 p53 依赖的 S 检查点与至少姐妹染色单体之间的同源重组联系起来。我们之前的研究表明，视网膜色素上皮中母体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，因此重组频率较高。受辐射精子受精后至少 11 至 12 天保持升高状态。事实上，受辐射精子受精的第 12 天胎儿的初级 MEF 中 SCE 的频率较高（正在准备中）。总而言之，我们为期 3 年的小鼠研究发现了一种新的 p53。依赖的S检查点及其在精子照射的胚胎中上调同源重组至少11至12天的功能。此外，他们还证明了DNA损伤记忆及其在延迟重组中的功能。为了研究其分子机制，我们还测试了裂殖酵母，看看是否可以诱导延迟和非靶向重组。 X射线照射后10个细胞周期代，上调重组的长度是细胞代数依赖性的，而不是绝对时间依赖性的，如温度变化实验所示。此外，这种上调重组的长度。这不是旁观者因素造成的，因为该现象不受培养基中自由基清除剂存在的影响，伴随着重组频率的上调，在辐射后观察到大约 10 代的 Rad22 焦点。粟酒裂殖酵母的记忆和延迟重组 Less

项目成果

期刊论文数量（43）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

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。

DOI：
发表时间：
2007
期刊：
影响因子：
0
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

Radiation induction of delayed recombination in S. pombe.

粟酒裂殖酵母延迟重组的辐射诱导。