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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

已知真核细胞在低启动剂量的辐射后具有增强辐射抵抗力的适应性反应。这种辐射适应性反应似乎提出了一种新的细胞防御机制。我们研究了蛋白激酶C(PKC)和丝裂原活化蛋白激酶(MAPK)在小鼠细胞辐射适应性反应表达中的作用。X射线照射后，PKC-a即被激活。低剂量X射线也激活了p38MAPK。P38MAPK的抑制剂SB203580和PKC的抑制剂Calphostin C可阻断p38MAPK的激活和对染色体畸变的抵抗。我们的结果表明，存在一种新的机制，通过PKC-a/p38MAPK反馈信号通路来协调调节对低剂量X射线的适应性反应。我们还利用X射线照射细胞的核提取液体外系统分析了dna中双链断裂的末端连接反应，因为dna中的双链断裂(Dsb)已经是…。更多地被认为是电离辐射引起的严重损伤，导致染色体异常、突变和细胞死亡。3Gy预照射细胞提取液(2cGy3Gy提取液)孵育5h后的末端连接效率高于3GyX射线照射细胞提取液(3Gy提取液)。根据突变质粒的频率估计其保真度或末端连接。虽然3Gy组与未照射细胞的末端连接保真度相当，但2cGy3Gy组的保真度远高于这两个组。我们进一步检测了回收的双链断裂错接的质粒DNA中的连接序列。所有错接的质粒都含有从断裂点到两侧至少缺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直接重复序列。未照射组、3Gy组和2cGy3Gy组的重复序列差异无统计学意义。2cGY+3Gy组的缺失大小往往比3Gy组的小。