Defining Relevant Targets of the DNA Dependent Protein Kinase

描述（由申请人提供）：DNA依赖性蛋白激酶（DNA-PK）在非同源末端连接（NHEJ）（真核生物中两种主要的双链断裂修复（DSBR）途径之一）中处于中心位置。DNA-PK识别双链断裂（DSB）并将其他因子靶向损伤部位。DNA-PK的酶活性对其在NHEJ中的功能至关重要;然而，迄今为止，DNA-PK的催化亚基（DNA-PKcs）本身是已知是其自身酶活性的功能相关靶标的唯一NHEJ因子。DNA-PKcs的自磷酸化发生在许多位点，并且在功能上是复杂的;不同的磷酸化事件具有不同的功能后果。两个主要簇内的磷酸化允许激酶调节（正向和负向）受损DNA对其他NHEJ因子以及其他DNA修复途径的接近，特别是其他主要DSBR途径，同源重组（HR）。NHEJ中的后续步骤需要额外的DNA-PK磷酸化事件，包括激酶解离和失活。该研究的前两个目标将探索1）DNA-PK如何调节DNA末端通路，2）定义和表征DNA修复过程中所需的其他DNA-PK磷酸化事件。第三个目标将集中在DNA-PK如何调节DSBR通路的选择，并在活体动物中描述这种选择的生物学后果。对于一种类型的双链断裂，没有选择;在VDJ重组期间由RAG内切核酸酶引入的DSB仅由NHEJ修复。初步数据表明，RAG复合物通过与DNA-PK相互作用将其DSB靶向NHEJ。第四个目标将描述RAG诱导的断裂是如何被限制在NHEJ pathway.有一个正在形成的共识，即DNA依赖性蛋白激酶（DNA-PK）在某些物种中比在其他物种中更严格地需要，并且似乎DNA-PK缺乏与人类生命不相容。了解这个大的复杂的功能，以协调DNA修复的非同源末端连接途径，并可能调节访问的DNA损伤的其他修复途径，应该提供的DNA-PK如何促进基因组稳定性的基础知识。

DESCRIPTION (provided by applicant): The DNA dependent protein kinase (DNA-PK) is central in non-homologous end joining (NHEJ), one of two major double strand break repair (DSBR) pathways in eukaryotes. DNA-PK recognizes double strand breaks (DSBs) and targets other factors to the site of damage. DNA-PK's enzymatic activity is critical to its function in NHEJ; however, to date, the catalytic subunit of DNA-PK (DNA-PKcs) itself is the only NHEJ factor known to be a functionally relevant target of its own enzymatic activity. Autophosphorylation of DNA-PKcs occurs on many sites, and is functionally complex; distinct phosphorylation events have distinct functional consequences. Phosphorylation within two major clusters allows the kinase to regulate (both positively and negatively) access of the damaged DNA to other NHEJ factors as well as to other DNA repair pathways, especially the other major DSBR pathway, homologous recombination (HR). Additional DNA-PK phosphorylation events are required for subsequent steps in NHEJ including kinase dissociation and inactivation. The first two aims of the proposed research will explore 1) how DNA-PK regulates DNA end access and 2) define and characterize other DNA-PK phosphorylation events that are required during DNA repair. The third aim will focus on how DNA-PK regulates DSBR pathway choice, and characterize the biologic consequence of this choice in living animals. For one type of double strand break, there is no choice; DSBs introduced during VDJ recombination by the RAG endonuclease are exclusively repaired by NHEJ. Preliminary data suggest that the RAG complex targets its DSBs to NHEJ by interaction with DNA-PK. The fourth aim will characterize how RAG induced breaks are restricted to the NHEJ pathway.There is an emerging consensus that the DNA dependent protein kinase (DNA-PK) is more stringently required in some species than in others, and it seems likely that DNA-PK deficiency is not compatible with human life. Understanding how this large complex functions to coordinate DNA repair by the non-homologous end joining pathway and potentially regulate access of DNA lesions to other repair pathways should provide fundamental knowledge of how DNA-PK promotes genomic stability.