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

项目总结/摘要 我们的应用集中在DNA依赖性蛋白激酶（DNA-PK），一种新的DNA修复因子， 确定的作用，RNA代谢和癌症治疗的目标，并将使用遗传，细胞生物学和单 分子方法来剖析DNA-PK在淋巴瘤和白血病发生和治疗中的作用。 基因组的不稳定性是人类癌症的标志。非同源末端连接（NHEJ）是DNA的主要结构 双链断裂（DSB）修复途径，是生理基因重排所必需的， 在发育中的淋巴细胞中致癌的染色体易位。DNA-PK，由KU 70-KU 80组成 异源二聚体（KU）和大催化亚基（DNA-PKcs），是一种NHEJ因子，对两个末端加工至关重要 (e.g.,发夹打开）和NHEJ期间的末端连接。DNA-PKcs抑制剂正处于癌症的I/IIa期临床试验中 疗法在NHEJ过程中，KU与DNA末端结合，招募并激活DNA-PKcs。DNA-PKcs缺失废除 Artemis内切核酸酶介导的末端加工而不消除末端连接。我们发现， 激酶死亡（KD）（DNA-PKcsKD/KD）消除末端连接而不影响末端加工， DNA-PKcs的保护作用由其自身的激酶活性调节。DNA-PKcsKD/KD小鼠中的末端加工 被ATM抑制剂阻断，表明终末加工需要DNA-PKcs蛋白，并且来自 体内DNA-PKcs或相关ATM激酶。DNA-PKcs是DNA-PK的最佳底物 也可以被ATM磷酸化。携带磷酸化缺陷型（DNA-PKcs 5A/5A）DNA-PKcs的小鼠 显示轻微的末端连接缺陷并且对ATM抑制敏感。因此，我们建议， KU结合的DNA，DNA-PK磷酸化调节末端加工，并最终释放DNA-PKcs， 许可证末端结扎。此外，我们发现Ku还可以指导DNA-PKcs在结构化RNA上的组装 (e.g., rRNA和snoRNA），其中磷酸化缺陷（DNA-PK 5A）或KD DNA-PKcs（DNA-PKcsKD/KD） 阻断rRNA加工、蛋白质翻译和红细胞生成，导致Trp 53依赖性骨髓衰竭。 这些发现揭示了DNA-PK在哺乳动物中的NHEJ独立作用。三分之二的DNA PKcsKD/KDTrp 53-/-小鼠死于核糖体应激诱导的髓性白血病，三分之一死于 IgH-Myc易位的淋巴瘤，突出了DNA-PK在肿瘤抑制中的关键作用。基于 这些和其他发现，我们假设DNA-PKcs激酶活性和自身磷酸化调节 DNA和RNA上DNA-PK的KU依赖性组装以抑制淋巴瘤和白血病的发生。到 为了验证这一点，我们将1）表征和比较KU和DNA-PK在RNA与DNA上动力学; 2）阐明KU- 耗尽影响人细胞中的RNA加工; 3）确定KU 80 C-末端的生理功能 结构域和尾部在淋巴瘤和白血病发生中的作用以及DNA-PKcs的募集和稳定。的 结果将揭示DNA-PK的RNA和DNA依赖功能的调节和功能， 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.