DNA-PKcs and PIDD interaction in DNA damage response

项目摘要/摘要 DNA依赖的蛋白激酶催化亚单位(DNA-PKcs)是经典的非蛋白激酶的关键调节因子。 DNA双链断裂修复与抗性的同源末端连接(NHEJ)途径 电离辐射(IR)。DNA-PKcs被Ku70/80异源二聚体募集到DSB末端形成DNA-PK 全酶启动NHEJ机制。DNA-PKcs在细胞对 复制压力。它与ATR(共济失调性毛细血管扩张症突变)协调并迅速被ATR磷酸化 和Rad3相关)，在停滞的复制分叉紫外线，尽管其机制还不是很清楚。 我们最近的工作集中在确定招募DNA-PKCC所需的球员(S)以使其陷入停滞 复制分叉。我们已经确定了PIDD(具有死亡结构域的P53诱导蛋白)，这是一种已知的细胞凋亡 DNA-PKcs需要组装PIDDosome复合体和激活Caspase-2的介体 对停滞的复制分叉的招募及其与ATR的关联。破坏DNA之间的相互作用- PKcs和PIDD不仅影响依赖ATR的DNA-PKcs在Thr2609簇的磷酸化，而且 还可减弱ATR信号通路和内S检查点。为了协助我们的调查，我们已经创建了 细胞株和表达DNA-PKcsPL突变蛋白的小鼠模型，该突变蛋白无法与PIDD相互作用。我们的结果 结果表明，DNA-PKcsPL细胞对紫外线和红外均高度敏感。根据这些初步调查结果，我们 假设PIDD，而不是Ku70/80异二聚体，介导DNA-PKcs募集到停滞的复制 分叉并促进其与ATR途径的关联。我们还假设PIDD促进DNA-PKcs 激酶活化和DSB修复。最后，我们假设DNA-PKcs与PIDD的相互作用将影响 细胞死亡调控与癌症发生。在这个项目中，我们将确定DNA- PKcs、PIDD和ATR在细胞对复制应激反应中的作用。我们的具体目标是：1)测试 PIDD在停滞复制叉处和IR诱导的DNA处调节DNA-PKcs激酶激活的假说 不受Ku70/80结合的损伤，2)测试DNA-PKcs正确需要PIDD的假设 在紫外线照射下停滞的复制分叉的功能，以及3)检验DNA-PKcs-PIDD的假设 关联性在DNA损伤和癌症发展过程中影响细胞命运的决定。

Project Summary/Abstract The DNA dependent protein kinase catalytic subunit (DNA-PKcs) is a key regulator of the canonical non- homologous end-joining (NHEJ) pathway for repair of DNA double strand breaks (DSBs) and resistance to ionizing radiation (IR). DNA-PKcs is recruited by the Ku70/80 heterodimer to the DSB ends to form the DNA-PK holoenzyme to initiate NHEJ mechanism. DNA-PKcs also plays an important role in cellular resistance to replication stress. It coordinates with and is rapidly phosphorylated by the ATR (ataxia telangiectasia mutated and Rad3 related) kinase at stalled replication forks upon UV, although the mechanism is not well understood. Our recent work has focused on identifying the player(s) required for the recruitment of DNA-PKcs to stalled replication forks. We have identified that PIDD (p53-induced protein with a death domain), a known apoptosis mediator for assembling the PIDDosome complex and Caspase-2 activation, is required for DNA-PKcs recruitment to stalled replication forks and its association with ATR. Disrupting the interaction between DNA- PKcs and PIDD not only compromised ATR dependent DNA-PKcs phosphorylation at the Thr2609 cluster but also attenuated the ATR signaling pathway and intra-S checkpoint. To assist our investigation, we have created cell lines and a mouse model expressing a DNA-PKcsPL mutant protein unable to interact with PIDD. Our results showed that DNA-PKcsPL cells were highly sensitive to both UV and IR. Based on these preliminary findings, we hypothesize that PIDD, but not the Ku70/80 heterodimer, mediates DNA-PKcs recruitment to stalled replication forks and promotes its association with the ATR pathway. We also hypothesize that PIDD facilitates DNA-PKcs kinase activation and DSB repair. Finally, we hypothesize that the interaction of DNA-PKcs with PIDD will affect cell death regulation and cancer development. In this project, we will determine the coordination between DNA- PKcs, PIDD, and ATR in the cellular response to replication stress. Our specific aims are: 1) To test the hypothesis that PIDD modulates DNA-PKcs kinase activation at stalled replication forks and at IR-induced DNA lesions that are not bound by Ku70/80, 2) To test the hypothesis that PIDD is required for DNA-PKcs to properly function at stalled replication forks upon UV irradiation, and 3) To test the hypothesis that the DNA-PKcs-PIDD association affects cell fate determination upon DNA damage and cancer development.