DNA-PKcs and PIDD interaction in DNA damage response

DNA 损伤反应中 DNA-PKcs 和 PIDD 相互作用

• 批准号: 10228547
• 负责人: Benjamin Ping-Chi Chen
• 金额: $ 37.06万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10228547
• 关键词: Affect; Apoptosis; Attenuated; B-Cell Lymphomas; CASP2 gene; Cancer Model; Catalytic Domain; Cell Culture Techniques; Cell Death; Cell Differentiation process; Cell Line; Cell Proliferation; Cell Survival; Cells; Complex; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA Repair Pathway; DNA lesion; DNA replication fork; DNA-PKcs; DNA-dependent protein kinase; Death Domain; Development; Double Strand Break Repair; G22P1 gene; Genome; Holoenzymes; In Vitro; Incidence; Inflammation; Investigation; Ionizing radiation; Liver; Malignant Neoplasms; Malignant neoplasm of liver; Mediating; Mediator of activation protein; Modeling; Mus; Mutation; Nonhomologous DNA End Joining; Normal Cell; Oncogene Activation; Oncogenes; Pathway interactions; Phosphorylation; Phosphotransferases; Play; Primary carcinoma of the liver cells; Process; Proteins; Radiation Tolerance; Radiation exposure; Reagent; Recovery; Regulation; Reporting; Resistance; Role; Signal Pathway; Steatohepatitis; TP53 gene; Testing; Tissues; UV carcinogenesis; Ultraviolet Rays; Work; ataxia telangiectasia mutated protein; base; biological adaptation to stress; cancer cell; cancer prevention; carcinogenesis; genome integrity; in vivo; male; mouse model; mutant; novel; preservation; prevent; recruit; regenerative; repaired; replication stress; response; senescence; tumorigenesis; ultraviolet irradiation

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.

项目摘要/摘要 DNA依赖性蛋白激酶催化亚基（DNA-PKC）是规范非 - 的关键调节剂 DNA双链断裂（DSB）修复的同源最终连接（NHEJ）途径 电离辐射（IR）。 DNA-PKC由KU70/80异二聚体募集到DSB末端以形成DNA-PK 全酶启动NHEJ机制。 DNA-PKC在细胞阻力中也起着重要作用 复制应力。它与ATR（共济失调突变的ata省 尽管该机理尚不清楚，但在紫外线上的复制叉处的RAD3相关）激酶。 我们最近的工作重点是确定招募DNA-PKC所需的玩家 复制叉。我们已经确定PIDD（p53诱导的具有死亡结构域的蛋白），一种已知的凋亡 DNA-PKC需要组装Piddosome复合物和caspase-2激活的介体 招募停滞的复制叉及其与ATR的关联。破坏DNA-之间的相互作用 PKC和PIDD不仅损害了Thr2609群集的ATR依赖性DNA-PKCS磷酸化，还会损害 还减弱了ATR信号通路和内部检查点。为了协助我们的调查，我们创建了 表达DNA-PKCSPL突变体蛋白无法与PIDD相互作用的细胞系和小鼠模型。我们的结果 表明DNA-PKCSPL细胞对UV和IR都高度敏感。根据这些初步发现，我们 假设PIDD，但不是KU70/80异二聚体，将DNA-PKCS募集介导了降低的复制 叉子并促进其与ATR途径的关联。我们还假设PIDD促进DNA-PKCS 激酶激活和DSB修复。最后，我们假设DNA-PKC与PIDD的相互作用会影响 细胞死亡调节和癌症发展。在这个项目中，我们将确定DNA-之间的协调 PKC，PIDD和ATR在对复制应力的细胞反应中。我们的具体目的是：1）测试 PIDD在停滞的复制叉和IR诱导的DNA处调节DNA-PKCS激酶激活的假设 不受KU70/80的约束的病变，以检验DNA-PKC需要PIDD正确的假设 紫外线照射时停滞的复制叉的功能，以及3）测试DNA-PKCS-PIDD的假设 关联会影响细胞命运对DNA损伤和癌症发展。