ROLE OF THE NUCLEAR MATRIX IN DOUBLE STRAND BREAK REPAIR

本项目提出的研究目标是测试 DNA-核基质对DNA组织作用假说 (NM)附着点是细胞反应的关键影响， 辐射引起的DNA损伤 为了验证这一假设，我们提出 下面的.首先，我们将识别特定蛋白质的变化 其构成不同放射敏感性细胞系的NM， 2D-PAGE、微测序、细胞内定位和DNA 结合力 其次，我们将描述差异 在遗传水平上， 通过差异mRNA显示，然后PCR介导扩增 与辐射抗性相关的序列。第三，我们将测试模型， 根据目前的数据，预测效率的差异， 修复双链DNA断裂。 测试这些模型将 包括测量DNA双链断裂的可修复性， 转录状态在DNA结构域中的功能， 断裂和相邻结构域由表征的NM分开 附着点。实验将利用核酸内切酶诱导的 特定基因的双链断裂，以评估 转录对这些断裂的修复。 第四，我们建议 开发改进的方法，以快速测定DNA损伤， 反映观察到的差异的核结构背景 在辐射敏感细胞和辐射抗性细胞之间， 临床肿瘤样本，以便上述原则可以在 人类肿瘤细胞。 通过完成这些具体的 aims将有助于增强对内在细胞的理解 放射敏感性及其与肿瘤放射可治愈性的关系。

The objective of the research proposed in this project is to test the hypothesis that DNA organization imposed by DNA-nuclear matrix (NM) attachment points is a key influence in cellular response to radiation-induced DNA damage. To test this hypothesis we propose the following. First, we will identify changes in specific proteins which constitute the NM of cell lines of differing radiosensitivity by 2D-PAGE, microsequencing, intracellular localization and DNA binding capacity. Second, we will characterize the differences between radioresistant and radiosensitive cell lines at the genetic level by differential mRNA display followed y PCR-mediated amplification of radioresistance-associated sequences. Third, we will test models, based on current data which predict differences in the efficiency of repair of double-strand DNA breaks. Testing these models will involve measuring the reparability of DNA double-strand breaks as a function of transciptional status within a DNA domain containing breaks and adjacent domains separated by characterized NM attachment points. Experiments will utilize endonuclease-induced double-stand breaks in specific genes to assess the effects of transcription on the repair of these breaks. Fourth, we propose to develop improved methods to rapidly assay DNA damage in the context of nuclear structure reflecting the observed differences between radiosensitive and radioresistant cells that are applicable to clinical tumor samples so that the above principles can be tested in human tumor cells. Insights gained by accomplishing these specific aims will contribute to an enhanced understanding of intrinsic cellular radiosensitivity and its relationship to tumor radiocurability.