HYPERTHERMIA AND THE FUNCTIONS OF HSP70

Ku自身抗原是一种DNA结合蛋白，由70 kDa（Ku70）组成。 和86-kDa（Ku80）亚基。最近的数据表明，Ku80在 DNA双链断裂的修复，以及Ku70抑制 热诱导hsp70表达。该项目的长期目标是 来理解细胞对压力反应的分子基础。 下一个赠款期的工作将侧重于机制和 Ku蛋白的功能方面，在调节细胞反应， 热冲击和电离辐射。有三个具体目标： 具体目标I侧重于Ku 抑制HSP 70的热诱导。我们要验证一个假设 从我们的初步研究中推断，Ku调节热量， 休克反应通过调节转录因子的结合， 它们各自的调控元件在启动子区的 hsp70基因。 通过体内基因组足迹法，研究了Ku对细胞内基因表达模式的影响。 蛋白结合的hsp70启动子，特别是其对 热休克转录激活因子HSF1和其他 转录因子与它们各自的结合位点的结合将是 测定Ku与特定元素结合的可能性 将测试hsp70基因的调节区。此外 到体内基因组足迹，突变将被引入到 将hsp70的启动子与报告基因构建体连接，以检测 对于顺式元件的存在，当突变时， Ku对报告基因热诱导抑制作用 表情 在具体目标II中，我们将研究结构和功能 Ku的结构域参与hsp70基因的调节 表情稳定和组成型表达的啮齿动物细胞系 将建立各种突变的Ku基因。热休克反应 以及野生型和突变型的生化特性 Ku亚单位将被检查。 在具体目标III中，我们建议建立纯合啮齿动物细胞 其中Ku70或Ku80的两个等位基因都被灭活的品系， 基因靶向诱变这些"双淘汰" Ku7O-/-和Ku8- /-细胞系将使我们能够研究的各种生理作用， Ku的各个亚基，并剖析其功能结构域， 参与细胞对热休克和电离的反应 辐射由于生物学上的重要性和 Ku的细胞作用，构建Ku 70-/-的意义， Ku80-/-线超出了阐明 Ku在hsp70基因调控和细胞敏感性中的作用 到X光。这些细胞系及其衍生物表达多种 突变Ku亚基可以直接用于任何突变Ku亚基的分析。 Ku的功能方面。胚胎干细胞的选择 这项工作进一步保证了这些研究可以很容易地扩展到 动物模型，通过构建基因敲除小鼠品系。

Ku autoantigen is a DNA binding protein consisting of 7O-kDa (Ku70) and 86-kDa (Ku80) subunits. Recent data have implicated Ku80 in the repair of DNA double strand breaks, and Ku70 in the suppression of heat-induced hsp70 expression. The long term goal of this project is to understand the molecular bases of the cellular response to stress. Work in the next granting period will focus on the mechanistic and functional aspects of Ku protein, in modulating cellular response to heat shock and to ioizing radiation. There are three specific aims: Specific Aim I focuses on the molecular mechanism(s) by which Ku suppresses heat-induction of hsp70. We will test a hypothesis inferred from our preliminary studies that Ku regulates the heat shock response by modulating the binding of transcription factors to their respective regulatory elements in the promoter region of the hsp70 gene. By in vivo genomic footprinting, the effects of Ku on the pattern of protein binding to the hsp70 promoter, especially its effects on the binding of the heat shock transcription activator HSF1 and other transcription factors to their respective binding sites will be determined. The possibility that Ku binds to a specific element in the regulatory region of the hsp70 gene will be tested. In addition to in vivo genomic footprinting, mutations will be introduced into the promoter of hsp70, linked to a reporter gene construct, to test for the presence of a cis-element(s), which when mutated abolishes the Ku-mediated suppression of heat-induction of reporter gene expression. In Specific Aim II we will study the structural and functional domains of Ku that are involved in the modulation of hsp70 gene expression. Rodent cell lines stably and constitutively expressing various mutant Ku genes will be established. The heat shock response of these cells and the biochemical properties of wild type and mutant Ku subunits will be examined. In Specific Aim III we propose to establish homozygous rodent cell lines in which both alleles of Ku70 or Ku80 are inactivated through gene-targeting mutagenesis. These "double knockout" Ku7O-/- and Ku8- /- cell lines will enable us to study various physiological roles of the individual subunits of Ku and to dissect the functional domains involved in the cellular response to heat shock and ionizing radiation. Because of the biological importance and the multiple cellular roles of Ku, the significance of constructing Ku70-/- and Ku80-/- lines extends beyond the immediate goal of elucidating the role of Ku in hsp70 gene regulation, and in the sensitivity of cells to x-rays. These cell lines and their derivatives expressing various mutant Ku subunits can be used directly in the analysis of any functional aspect of Ku. The choice of embryonic stem cells for this work further assures that such studies can be readily extended to animal models, through the construction of knockout mice strains.