ROLE OF THE NUCLEAR MATRIX IN DOUBLE STRAND BREAK REPAIR

这个项目中提出的研究的目的是测试 DNA-核基质强加的DNA组织假说 (NM)附着点是细胞响应的关键影响因素 辐射引起的DNA损伤。为了检验这一假设，我们提出了 以下是。首先，我们将确定特定蛋白质的变化 它们通过以下方式构成不同辐射敏感性的细胞系的NM 2D-PAGE、微测序、细胞内定位和DNA 结合能力。第二，我们将描述不同之处 辐射抗性和辐射敏感细胞系在基因水平上的差异 经差异显示后再经聚合酶链式反应进行扩增 与辐射抗性相关的序列。第三，我们将测试模型， 基于目前的数据，这些数据预测了两种药物的效率差异 修复双链DNA断裂。测试这些型号将 包括测量DNA双链断裂的可修复性作为 DNA结构域中转录状态的作用 由特征NM分隔的断点和相邻域 附着点。实验将利用核酸内切酶诱导的 在特定基因上的双立场突变来评估 关于修复这些断裂的转录。第四，我们建议 开发改进的方法来快速检测DNA损伤 反映观察到的差异的核结构的背景 对辐射敏感和抵抗辐射的细胞之间的关系 以便在临床肿瘤标本中检验上述原理 人类肿瘤细胞。通过完成这些具体任务而获得的见解 AIMS将有助于加深对内在细胞的理解 放射敏感性及其与肿瘤放射治疗能力的关系。

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.