INTERACTIONS OF DNA-PK WITH THE TRANSCRIPTION APPARATUS

对双链DNA断裂的反应能力在所有活细胞中都是至关重要的。在真核生物中，DNA依赖性蛋白激酶（DNA- pk）在双链DNA断裂的修复和相关的信号传导过程中发挥作用。有证据表明，DNA- PK部分通过与RNA聚合酶II和转录因子的功能相互作用发挥其信号功能。所提出的研究的目标是更详细地了解这些相互作用并评估其生物学意义。本项目分为两部分。第一部分涉及DNA-PK和一般转录机制之间的关系。这些研究使用缺乏DNA-PK （Ku蛋白）或催化亚基（DNA-PKcs）的调控成分的突变细胞。在体外实验中，突变细胞的核提取物在进行多轮转录的能力上存在缺陷。一系列的实验将进行，以了解这种转录缺陷的潜在机制。实验还将测试DNA- pk与一般转录机制的相互作用是否允许DNA- pk调节体内响应DNA损伤的全局转录水平。该建议的第二部分涉及DNA-PK和启动子特异性转录激活子蛋白之间的关系。拟议的实验旨在了解哪些候选因子实际上在体内与DNA-PK相互作用。进一步的实验将包括对辐射损伤细胞中DNA-PK酶特性变化的表征。可能存在新的调控机制，允许起源于DNA断裂的信号被放大并在整个细胞核中传播。DNA- pk在许多涉及DNA断裂和DNA末端的重要过程中起着关键作用。本提案中的实验将提供有关DNA-PK和转录装置之间发生的信号相互作用的明确信息。

The ability to respond to double-strand DNA breaks is of fundamental importance in all living cells. In eukaryotes, the DNA-dependent protein kinase (DNA-PK) plays a role in both the repair of double-strand DNA breaks and in associated signaling processes. Evidence suggests that DNA- PK exerts it signaling functions, in part, through functional interactions with RNA polymerase II and transcription factors. The goal of the proposed studies is to understand these interactions in more detail and to evaluation their biological significance. This project is divided into two parts. The first deals with the relationship between DNA-PK and the general transcription machinery. These studies use mutant cells that lack either the regulatory component of DNA-PK (the Ku protein) or the catalytic subunit (DNA-PKcs). Nuclear extracts from the mutant cells are defective in their ability to carry out multiple rounds of transcription in an in vitro assay. A series of experiments will be undertaken in order to understand the underlying mechanisms of this transcription defect. Experiments will also be performed to test whether interaction of DNA-PK with the general transcription machinery allows DNA-PK to modulate global levels of transcription in vivo in response to DNA damage. A second part of the proposal deals with the relationship between DNA-PK and promoter-specific transcriptional activator proteins. Proposed experiments are directed toward understanding which of the many candidate factors actually interact with DNA-PK in vivo. Additional experiments will involve characterization of changes in the enzymatic properties of DNA-PK in cells subjected to radiation injury. Novel regulatory mechanisms may exist that allow a signal originating at a DNA break to be amplified and propagated throughout the nucleus. DNA-PK has emerged as a key player in a number of important processes involving DNA breaks and DNA ends. Experiments in this proposal will provide definitive information about signaling interactions that occur between DNA-PK and the transcription apparatus.