INTERACTIONS OF DNA-PK WITH THE TRANSCRIPTION APPARATUS

对双链DNA断裂做出反应的能力在所有活细胞中都是至关重要的。在真核生物中，DNA依赖的蛋白激酶(DNA-PK)在双链DNA断裂的修复和相关的信号过程中都发挥着作用。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来发挥其信号功能。拟议研究的目标是更详细地了解这些相互作用，并评估它们的生物学意义。该项目分为两个部分。第一部分是DNA-PK与一般转录机制的关系。这些研究使用的突变细胞既没有DNA-PK(Ku蛋白)的调节成分，也没有催化亚基(DNA-PKcs)。突变细胞的核提取液在体外实验中进行多轮转录的能力存在缺陷。为了了解这种转录缺陷的潜在机制，将进行一系列实验。还将进行实验，以测试DNA-PK与一般转录机制的相互作用是否允许DNA-PK在体内调节整体转录水平，以应对DNA损伤。该提案的第二部分涉及DNA-PK和启动子特异性转录激活蛋白之间的关系。拟议的实验旨在了解许多候选因素中的哪些实际上在体内与DNA-PK相互作用。其他实验将包括表征DNA-PK在受到辐射损伤的细胞中的酶性质的变化。可能存在新的调节机制，允许来自DNA断裂的信号被放大并在整个细胞核中传播。DNA-PK在涉及DNA断裂和DNA末端的许多重要过程中扮演着关键角色。这项提议中的实验将提供关于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.