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

已知真核细胞具有自适应反应，可在低启动剂量的辐射剂量后增强放射性反应。这种放射性的反应似乎提出了一种新型的细胞防御机制。我们研究了蛋白激酶C（PKC）和有丝分裂原激活的蛋白激酶（MAPK）在培养的小鼠细胞中放射性适应反应表达中的作用。 X辐照后立即激活PKC-A。低剂量X射线还激活了p38 MAPK。 p38 MAPK的激活和对染色体像差形成的抗性被p38 mapk的抑制剂SB203580和PKC的抑制剂Calphostin C阻断。我们的结果表明，存在一种新型机制，用于通过PKC-A/P38 MAPK反馈信号通路的Nexus协调对低剂量X射线的适应性反应。我们还使用体外系统与暴露于X射线的细胞的核提取物中分析了DNA中双链断裂的最终结合反应，因为DNA中的双链断裂（DSB）的核酸提取物已成为……更具EN的实现，它是通过电离导致辐射导致的临界病变来实现的，导致呈染色体的辐射，突变，突变和细胞死亡。孵育5小时（2CGY+3GY提取物）后，提取物中提取物的最终结合效率高于暴露于3GY X射线（3GY提取物）的细胞中的提取物。还从突变质粒的频率中估计了保真度或最终连接。尽管3GY提取物的最终结合保真度和未辐照的细胞的提取物是可比的，但2CGY+3GY提取物的保真度远高于这些提取物。我们进一步检查了回收的质粒DNA中的连接序列，并错过了双链断裂。所有错过的质粒都包含从断裂点到两侧缺少几个核苷酸的缺失。在所有接线点都发现了几个核苷酸的直接重复。通过未辐照的提取物，3GY提取物和2CGY+3GY提取物在重复序列之间未观察到显着差异。 2CGY+3GY提取物的删除尺寸往往比3Gy提取物小的尺寸要小