The role of DNA-PKcs in DNA repair, lymphocyte development, RNA metabolism and tumor suppression

项目摘要/摘要 我们的应用重点是DNA依赖的蛋白激酶(DNA-PK)，这是一种新的DNA修复因子 确定了在RNA新陈代谢中的作用和癌症治疗的目标，并将利用遗传学、细胞生物学和单一 分析DNA-PK在淋巴瘤和白血病发生和治疗中作用的分子方法。 基因组不稳定是人类癌症的标志。非同源末端连接(NHEJ)是一种主要的DNA 双链断裂(DSB)修复途径，是生理基因重排和 发育中淋巴细胞的致癌染色体易位。DNA-PK，由KU70-KU80组成 异源二聚体(KU)和大催化亚基(DNA-PKcs)是NHEJ因子，对两者的末端加工都至关重要 (例如，发夹打开)和在NHEJ期间的末端结扎。DNA-PKcs抑制剂正在进行癌症的I/IIa期临床试验 心理治疗。在NHEJ期间，KU与DNA末端结合，招募并激活DNA-PKcs。DNA-PKcs的丢失 Artemis内切酶介导的末端加工而不取消末端连接。我们向大家展示了 激酶死亡(Kd)(DNA-PKcsKD/KD)在不影响末端加工的情况下取消末端连接，揭示末端- DNA-PKcs的保护作用，受其自身的激酶活性调节。DNA-PKcsKD/KD小鼠的末端加工 被ATM抑制阻断，表明末端处理需要DNA-PKcs蛋白，而来自 DNA-PKcs或体内相关的ATM激酶。DNA-PKcs是dna-pk最具特性的底物。 也可以被自动取款机磷酸化。携带(DNA-PKcs5A/5A)DNA-PKcs的小鼠 表现出轻微的末端连接缺陷，对ATM抑制敏感。因此，我们建议，一旦组装在 Ku结合的DNA，DNA-PK的磷酸化调节末端加工，并最终将DNA-PKcs释放到 牌照结扎结束。此外，我们还发现Ku还可以指导dna-pkcs在结构rna上的组装。 (例如，rRNA和snoRNA)，其中磷酸化缺陷(DNA-PK5A)或KD DNA-PKcs(DNA-PKcsKD/KD) 阻止rRNA加工、蛋白质翻译和红细胞生成，导致Trp53依赖的骨髓衰竭。 这些发现揭示了DNA-PK在哺乳动物中的非NHEJ独立作用。以及三分之二的DNA- PKcsKD/KDTrp53-/-小鼠死于核糖体应激诱导的髓系白血病，三分之一死于 淋巴瘤伴有IgH-Myc易位，突出了DNA-PK在肿瘤抑制中的关键作用。基于 这些和其他的发现，我们假设dna-pkcs的活性和自动磷酸化调节。 Ku依赖的DNA-PK在DNA和RNA上的组装抑制淋巴瘤和白血病的发生。至 测试这一点，我们将1)表征和比较KU和DNA-PK在RNA和DNA上的动力学；2)阐明KU-PK是如何 耗竭对人类细胞RNA加工的影响；3)确定KU80C-末端的生理功能 淋巴瘤和白血病发生中的结构域和尾部以及DNA-PKcs的募集和稳定。这个 结果将揭示DNA-PK的RNA和DNA依赖功能的调节和功能，这是必不可少的 KU在人类细胞中的作用，以及DNA-PK抑制的广泛影响。

PROJECT SUMMARY/ABSTRACT Our application focus on DNA-dependent protein kinase (DNA-PK), a DNA repair factor with newly identified role in RNA metabolism and a target of cancer therapy, and will use genetic, cell biology and single molecule approaches to dissect the role of DNA-PK during lymphoma and leukemia-genesis and therapy. Genomic instability is the hallmark of human cancer. The Non-Homologous End-Joining (NHEJ) is a major DNA double-strand breaks (DSBs) repair pathway and is required for physiological gene-rearrangements and oncogenic chromosomal translocations in developing lymphocytes. DNA-PK, composed of KU70-KU80 heterodimer (KU) and the large catalytic subunit (DNA-PKcs), is a NHEJ factor critical for both end-processing (e.g., hairpin opening) and end-ligation during NHEJ. DNA-PKcs inhibitors is in phase I/IIa clinical trials for cancer therapy. During NHEJ, KU binds to DNA ends, recruits and activates DNA-PKcs. Loss of DNA-PKcs abrogate Artemis endonuclease mediated end-processing without abolishing end-ligation. We showed that expression of kinase-dead (KD) (DNA-PKcsKD/KD) abrogates end-ligation without affecting end-processing, uncovering an end- protection role of DNA-PKcs that is regulated by its own kinase activity. End-processing in DNA-PKcsKD/KD mice is blocked by ATM inhibition, indicating end-processing requires DNA-PKcs protein, and the kinase activity from either DNA-PKcs or the related ATM kinase in vivo. DNA-PKcs is the best characterized substrate of DNA-PK and can also be phosphorylated by ATM. Mice carrying phosphorylation-deficient (DNA-PKcs5A/5A) DNA-PKcs display mild end-ligation defects and are sensitive to ATM inhibition. Thus, we propose that once assembled on KU-bound DNA, DNA-PK phosphorylation regulates end-processing and eventually the release of DNA-PKcs to licence end-ligation. Moreover, we found that Ku can also direct the assembly of DNA-PKcs on structured RNA (e.g., rRNA and snoRNA), where phosphorylation defective (DNA-PK5A) or KD DNA-PKcs (DNA-PKcsKD/KD) blocks rRNA processing, protein translation, and erythropoiesis, leading to Trp53-dependent bone marrow failure. These findings uncovered a NHEJ-independent role of DNA-PK in mammals. And two-third of DNA- PKcsKD/KDTrp53-/- mice succumbed to ribosomal stress induced myeloid leukemia and one-third died of lymphomas with IgH-Myc translocations, highlighting the critical role of DNA-PK in tumor suppression. Based on these and other findings, we hypothesize that DNA-PKcs kinase activity and auto-phosphorylation regulates KU-dependent assembly of DNA-PK on DNA and RNA to suppress lymphoma and leukemia genesis. To test this, we will 1) characterize and compare KU and DNA-PK dynamics on RNA vs DNA; 2) elucidate how KU- depletion impact RNA processing in human cells; 3) determine the physiological function of KU80 C-terminal domain and tail in lymphoma and leukemia genesis and the recruitment and stabilization of DNA-PKcs. The results will reveal the regulation and function of the RNA & DNA dependent function of DNA-PK, the essential role of KU in human cells, and the broad impacts of DNA-PK inhibition.