Transgenic Mouse for DNA Damage Sensing and Signaling

当哺乳动物细胞暴露在电离辐射(IR)中时，一系列分子事件被触发。这些事件包括DNA损伤识别、损伤信号、DNA修复和细胞周期控制。虽然已经鉴定了一些参与IR反应的基因和基因产物，但这些过程是如何协调的还不清楚。本研究的目的是了解三种主要的DNA损伤传感和信号转导蛋白之间的相互关系：DNA-PK(DNA依赖蛋白激酶)、ATM(共济失调毛细血管扩张症突变)和Nbs1(奈梅亨断裂综合征基因产物突变)。我们假设这些DNA损伤传感和信号蛋白在时间和/或空间上是协调的，以响应哺乳动物细胞中的DNA损伤。为了验证这一假设，我们计划在原代小鼠胚胎成纤维细胞(MEF)和转基因小鼠细胞系中检测这些蛋白质之间的功能关系(S)，这些细胞系对这三种蛋白质的不同组合是单基因敲除或双基因敲除(KO)。我们已经在实验室中产生或获得了Ku70、Ku80、DNA-PKcs、Ku80/DNA-PKcs和ATM基因敲除的小鼠。为了避免Nbs1基因敲除和ATM/DNA-PKcs双重敲除对胚胎和躯体的致命影响，我们计划使用我们实验室设计的一步Tet-off/CreLoxP策略来产生DNA-PKcs和Nbs1的条件敲除。利用这些基因敲除和/或条件性基因敲除小鼠，我们计划阐明1)DNA-PK的Ku成分在DNA损伤调节ATM激酶激活中的作用，2)ATM和DNA-PK的单独和重叠的作用，以及3)NBS1在DNA断裂的感知、信号传递和修复中的作用。从体外和体内对单基因敲除和双基因敲除小鼠的研究获得的初步结果为我们提供了对这些机制的见解。我们建议追踪这些线索，以更清楚地了解DNA-PK、ATM和Nbs1之间的协调机制。

A cascade of molecular events is triggered in mammalian cells upon exposure to ionizing radiation (IR). These events include DNA damage recognition, damage signaling, DNA repair, and cell cycle control. Although a number of genes and gene products involved in IR responses have been identified, it is not clear how these processes are coordinated. The purpose of this proposal is to understand the inter-relationship among three major DNA damage sensing and signaling proteins: DNA-PK (DNA- dependent protein kinase), ATM (Ataxia Telangiectasia Mutated), and Nbs1 (the gene product mutated in Nijmegen breakage syndrome). We hypothesize that these DNA damage sensing and signaling proteins are coordinated in a temporal and/or spatial fashion in response to DNA damage in mammalian cells. To test this hypothesis, we plan to examine the functional relationship(s) among these proteins in primary mouse embryonic fibroblasts (MEFs) and cell lines derived from transgenic mice that are single or double knockouts (KO) for various combinations of these three proteins. We have generated in the lab or have acquired mice that are knockouts for Ku70, Ku80, DNA-PKcs, Ku80/DNA-PKcs, and ATM. To avoid embryonic and somatic lethality for Nbs1 knockout and ATM/DNA-PKcs double knockout, we plan to generate conditional knockouts for DNA-PKcs and Nbs1 using a novel one-step Tet-Off/CreLoxP strategy designed in our lab. Using these knockout and/or conditional knockout mice, we plan to elucidate 1) the role of the Ku component of DNA-PK in the modulation of ATM kinase activation by DNA damage, 2) the individual and overlapping roles of ATM and DNA-PK, and 3) the role of Nbs1 in the sensing, signaling and repair of DNA breaks. Preliminary results obtained from in vitro as well as from in vivo studies on single and double knockout mice have provided us with insights into these mechanisms. We propose to follow up these leads to obtain a clearer picture of the coordinating mechanisms between DNA-PK, ATM, and Nbs1.