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Analysis of repair of double-strand breaks in DNA in the process of radioadaptive response using in vitro system

体外系统分析放射适应性反应过程中DNA双链断裂的修复

基本信息

批准号：
09680519
负责人：
TACHIBANA Akira
金额：
$ 2.75万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09680519/
关键词：
radioadaptive response repair of double-strand breaks in DNA cellular signal transduction p53 gene protein kinase C MAP kinase non-homologous end-joining

项目摘要

Eukaryotic cells are known to have an adaptive response that enhances radioresistance after a low priming dose of radiation. This radioadaptive response seems to present a novel cellular defense mechanism. We studied the role of protein kinase C(PKC) and mitogen-activated protein kinase (MAPK) in the expression of radioadaptive response in cultured mouse cells. PKC-a was activated immediately after X-irradiation. Low dose X-rays also activated the p38 MAPK. The activation of p38 MAPK and resistance to chromosome aberration formation were blocked by SB203580, an inhibitor of p38 MAPK, and Calphostin C, an inhibitor of PKC. Our results indicate the presence of a novel mechanism for coordinated regulation of adaptive response to low-dose X-rays by a nexus of PKC-a/p38 MAPK feedback signaling pathway. We also analyzed the end-joining reaction of double-strand breaks in DNA using in vitro system with nuclear extracts from cells exposed to X-rays, as the double-strand break(dsb)in DNA has be … More en implicated as the critical lesion induced by ionizing radiation leading to chromosome aberrations, mutations and cell death. The efficiency of end-joining by the extract from preexposed cells challenged with 3Gy after incubation for 5 hours (2cGy+3Gy extract) was higher than that by the extract from cells exposed to 3Gy X-rays (3Gy extract). The fidelity or end-joining was also estimated from the frequency of mutant plasmids. Although the end-joining fidelities of the 3Gy extract and the extract from unirradiated cells were comparable, the fidelity of the 2cGy+3Gy extract was much higher than these. We further examined the junction sequences in the recovered plasmid DNA with mis-rejoining of double-strand breaks. All mis-rejoined plasmids contained deletions missing at least several nucleotides from the break point to both sides. Short direct repeats of several nucleotides were found at all junction points. No significant differences were observed among the repeat sequences by the unirradiated extract, 3Gy extract and 2cGy+3Gy extract. The sizes of deletions by the 2cGy+3Gy extract tended to be smaller than these by the 3Gy extract Less

已知真核细胞具有适应性反应，可在低剂量辐射后增强放射抗性。这种放射适应性反应似乎提出了一种新颖的细胞防御机制。我们研究了蛋白激酶 C (PKC) 和丝裂原激活蛋白激酶 (MAPK) 在培养小鼠细胞中放射适应性反应表达中的作用。 X射线照射后PKC-a立即被激活。低剂量 X 射线也激活了 p38 MAPK。 p38 MAPK 的激活和对染色体畸变形成的抵抗被 p38 MAPK 抑制剂 SB203580 和 PKC 抑制剂 Calphostin C 阻断。我们的结果表明存在一种新机制，通过 PKC-a/p38 MAPK 反馈信号通路的联系来协调调节对低剂量 X 射线的适应性反应。我们还使用体外系统和暴露于 X 射线的细胞核提取物分析了 DNA 双链断裂的末端连接反应，因为 DNA 中的双链断裂 (dsb) 被认为是电离辐射引起的严重损伤，导致染色体畸变、突变和细胞死亡。孵育5小时后用3Gy激发的预暴露细胞提取物（2cGy+3Gy提取物）的末端连接效率高于暴露于3Gy X射线的细胞提取物（3Gy提取物）的末端连接效率。保真度或末端连接也可以根据突变质粒的频率来估计。尽管 3Gy 提取物和未辐照细胞提取物的末端连接保真度相当，但 2cGy+3Gy 提取物的保真度远高于这些。我们进一步检查了回收的质粒 DNA 中的连接序列，以及双链断裂的错误重新连接。所有错误重新连接的质粒都包含从断裂点到两侧至少丢失几个核苷酸的缺失。在所有连接点都发现了几个核苷酸的短同向重复。未辐照提取物、3Gy提取物和2cGy+3Gy提取物的重复序列之间没有观察到显着差异。 2cGy+3Gy 提取物的缺失大小往往小于 3Gy 提取物的缺失大小更少