Defining Relevant Targets of the DNA Dependent Protein Kinase

描述(申请人提供)：DNA依赖蛋白激酶(DNA-PK)是真核生物中两个主要的双链断裂修复(DSBR)途径之一的非同源末端连接(NHEJ)的核心。DNA-PK识别双链断裂(DSB)，并针对损伤部位的其他因素。DNA-PK的酶活性对其在NHEJ中的功能至关重要；然而，到目前为止，DNA-PK的催化亚单位(DNA-PKcs)本身是唯一已知的与其自身酶活性相关的NHEJ因子。DNA-PKcs的自磷酸化发生在许多位点上，并且功能复杂；不同的磷酸化事件具有不同的功能后果。两个主要簇中的磷酸化允许该激酶调节(正向和负向)受损的DNA对其他NHEJ因子以及其他DNA修复途径的访问，特别是另一条主要的DSBR途径-同源重组(HR)。NHEJ的后续步骤还需要额外的DNA-PK磷酸化事件，包括激酶解离和失活。这项拟议研究的前两个目标将探索1)DNA-PK如何调节DNA末端通路和2)定义和表征DNA修复过程中需要的其他DNA-PK磷酸化事件。第三个目标将集中在DNA-PK如何调节DSBR途径的选择，并表征这种选择在活动物中的生物学后果。对于一种类型的双链断裂，没有选择；在VDJ重组过程中由RAG内切酶引入的DSB是由NHEJ独家修复的。初步数据表明，RAG复合体通过与DNA-PK相互作用将其DSB靶向NHEJ。第四个目标将描述RAG诱导的断裂是如何限制在NHEJ途径上的。一个新的共识是，DNA依赖的蛋白激酶(DNA-PK)在一些物种中比在另一些物种中需要更严格，而且DNA-PK缺陷似乎与人类的生命不相容。了解这个大型复合体如何通过非同源末端连接途径协调DNA修复，并潜在地调节DNA损伤进入其他修复途径，应该为DNA-PK如何促进基因组稳定性提供基础知识。

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.