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）是真核生物中两种主要双链断裂修复（DSBR）途径之一的非同源末端连接（NHEJ）的中心。DNA-PK识别双链断裂（DSBs）并将其他因素靶向到损伤部位。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）本身是唯一已知的NHEJ因子是其自身酶活性的功能相关靶标。DNA-PKcs的自磷酸化发生在许多位点，功能复杂；不同的磷酸化事件具有不同的功能后果。两个主要簇内的磷酸化允许激酶（积极和消极地）调节受损DNA进入其他NHEJ因子以及其他DNA修复途径，特别是另一个主要的DSBR途径，同源重组（HR）。NHEJ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。提出的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通路选择，并描述这种选择在活体动物中的生物学后果。对于一种类型的双股断裂，没有选择；由RAG内切酶在VDJ重组过程中引入的dsb仅由NHEJ修复。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诱导的断裂如何局限于NHEJ途径。DNA依赖性蛋白激酶（DNA- pk）在某些物种中比在其他物种中更被严格要求，似乎DNA- pk缺乏可能与人类的生命不相容。了解这个大复合体如何通过非同源末端连接途径协调DNA修复，并可能调节DNA损伤进入其他修复途径，将为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.