DNA-PKCS Phosphorylation in DNA Repair and Chromosomal Translocations

DNA 修复和染色体易位中的 DNA-PKCS 磷酸化

• 批准号: 8975763
• 负责人: Shan Zha
• 金额: $ 36.6万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-12-01 至 2019-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8975763
• 关键词: Address; Animal Model; B-Cell Lymphomas; B-Lymphocytes; C-terminal; Catalytic Domain; Cells; Chromosomal translocation; Cleaved cell; Code; DNA; DNA Damage; DNA Repair; DNA-PKcs; DNA-dependent protein kinase; Data; Defect; Development; Distal; Embryo; Embryonic Development; Excision; Future; G22P1 gene; Genetic Recombination; Genomic Instability; Goals; Health; Human; Immunoglobulin Class Switching; Immunoglobulin Genes; Immunoglobulin Switch Recombination; Immunoglobulins; In Vitro; KRP protein; Knock-in Mouse; Lead; Ligation; Lymphocyte; Lymphoma; Lymphomagenesis; Malignant lymphoid neoplasm; Mediating; Modification; Mus; Mutation; Nonhomologous DNA End Joining; Oncogenes; Oncogenic; Pathway interactions; Patients; Phosphorylation; Phosphorylation Site; Phosphotransferases; Process; Protein Kinase; Proteins; Radiation; Recurrence; Regulation; Reporting; Residual state; Risk; Role; Sequence Analysis; Site; Structure; TP53 gene; Testing; Tumor Suppression; V(D)J Recombination; artemis; ataxia telangiectasia mutated protein; base; cancer initiation; cancer therapy; chemotherapy; endonuclease; in vivo; leukemia/lymphoma; mouse model; programs; repaired; seal; tumor

DESCRIPTION (provided by applicant): Recurrent oncogenic translocations involving Ig or TCR loci characterize human lymphoid malignancies. These translocations often derived from mis-repaired DNA double stand breaks (DSBs) generated during normal lymphocyte development. Availability of free DNA end, proper end-processing and the mis-joining of distal DNA ends form the three essential steps for chromosomal translocations. Developmental DSBs in lymphocytes are normally repaired by the non-homologous end-joining (NHEJ) pathway with help from the alternative-end joining (A-EJ) pathway(s) that preferentially use micro-homology (MH) at the junctions. NHEJ is also responsible for end-processing, such as opening the hairpin ends at Ig/ TCR loci generated during V(D) recombination. Sequence analyses revealed that NHEJ and A-EJ also mediate the "mis"-repairs that generate translocations. Thus understanding the mechanism and regulation of NHEJ and A-EJ have broad implications in lymphomagenesis. DNA-PKcs is the catalytic subunit of the DNA-dependent protein-kinase (DNA-PK), a NHEJ factor and a PI3K related protein kinase. In the absence of DNA-PKcs, direct end-ligation is largely normal, but hairpin opening-a form of end-processing required for sealed hairpin ends before ligation, is completely blocked. Here we report that in a knockin mouse model expressing the kinase-dead (KD) form of DNA-PKcs alone (DNA-PKcsKD/KD), direct end-ligation is completely blocked, leading to embryonic lethality similar to core NHEJ deficient (e.g.Lig4-/- ) mice. Co-deletion of Ku70 that is necessary for the recruitment of DNA-PKcs to DNA, rescues the embryonic development of DNA-PKcsKD/KD mice, indicating that DNA-PKcs protein regulates end-ligation at DNA ends. Despite end-ligation defects, DNA-PKcsKD/KD cells open hairpin normally, but only in the presence of normal ATM kinase activity, revealing a previous unrecognized role of ATM in hairpin opening. Finally in contrast to frequent A-EJ mediated IgH-Myc translocations and aggressive lymphomas in other NHEJ/p53 double deficient mice, lymphomas are rare in DNA-PKcsKD/KDp53-/- mice despite the severe genomic instability, indicating potentially defects in A-EJ. DNA-PKcs is auto-phosphorylated and phosphorylated by ATM upon DSBs. Based on these findings, we hypothesize that DNA-PKcs phosphorylation regulates hairpin opening (Aim1), end-ligation (Aim 2) and A-EJ (Aim3) to suppress lymphomagenesis. Aim1 will address how redundant phosphorylation of DNA-PKcs and Artemis by ATM and DNA-PKcs promotes hairpin opening. Aim 2 proposes to test the hypothesis that autophosphorylation of DNA- PKcs at the DNA ends is necessary for ends-ligation by identifying functional relevant autophosphorylation sites on DNA-PKcs. Aim 3 will test the hypotheses that DNA-PKcs KD protein physically blocks end-resection and suppress A-EJ and oncogenic translocation by characterizing class switch recombination (mediated by A- EJ and NHEJ) and IgH-myc oncogenic translocations in DNA-PKcsKD/KD cells and mice. Together these studies will determine the role of DNA-PKcs phosphorylation in DNA repair and translocations. In the future, DNA- PKcs phosphorylation will provide an attractive target to regulate NHEJ and A-EJ for cancer treatments.

描述（由申请人提供）：涉及 Ig 或 TCR 位点的复发性致癌易位是人类淋巴恶性肿瘤的特征。这些易位通常源自正常淋巴细胞发育过程中产生的错误修复的 DNA 双链断裂 (DSB)。游离 DNA 末端的可用性、正确的末端加工和远端 DNA 末端的错误连接构成了染色体易位的三个基本步骤。淋巴细胞中发育中的 DSB 通常通过非同源末端连接 (NHEJ) 途径在选择性末端连接 (A-EJ) 途径的帮助下进行修复，该途径优先在连接处使用微同源性 (MH)。 NHEJ 还负责末端加工，例如打开 V(D) 重组过程中生成的 Ig/TCR 位点处的发夹末端。序列分析显示 NHEJ 和 A-EJ 也介导产生易位的“错误”修复。因此，了解 NHEJ 和 A-EJ 的机制和调节对于淋巴瘤发生具有广泛的意义。 DNA-PKcs 是 DNA 依赖性蛋白激酶 (DNA-PK)、NHEJ 因子和 PI3K 相关蛋白激酶的催化亚基。在缺乏 DNA-PKc 的情况下，直接末端连接在很大程度上是正常的，但发夹打开（连接前密封发夹末端所需的一种末端处理形式）被完全阻断。在这里，我们报道，在仅表达激酶死亡（KD）形式的DNA-PKcs（DNA-PKcsKD/KD）的敲入小鼠模型中，直接末端连接被完全阻断，导致类似于核心NHEJ缺陷的胚胎致死性（例如，DNA-PKcsKD/KD）。 Lig4-/-) 小鼠。 Ku70 的共缺失是 DNA-PKcs 募集至 DNA 所必需的，可挽救 DNA-PKcsKD/KD 小鼠的胚胎发育，表明 DNA-PKcs 蛋白调节 DNA 末端的末端连接。尽管存在末端连接缺陷，DNA-PKcsKD/KD 细胞仍能正常打开发夹，但前提是存在正常的 ATM 激酶活性，这揭示了 ATM 在发夹打开中先前未被认识的作用。最后，与其他 NHEJ/p53 双缺陷小鼠中频繁的 A-EJ 介导的 IgH-Myc 易位和侵袭性淋巴瘤相比，尽管基因组严重不稳定，但淋巴瘤在 DNA-PKcsKD/KDp53-/- 小鼠中很少见，这表明 A- 中存在潜在缺陷。 EJ。 DNA-PKcs 会自动磷酸化，并通过 DSB 上的 ATM 进行磷酸化。基于这些发现，我们假设 DNA-PKcs 磷酸化调节发夹开放 (Aim1)、末端连接 (Aim 2) 和 A-EJ (Aim3) 以抑制淋巴瘤发生。 Aim1 将解决 ATM 和 DNA-PKcs 对 DNA-PKcs 和 Artemis 的冗余磷酸化如何促进发夹打开。目标 2 提议通过识别 DNA-PKcs 上功能相关的自磷酸化位点来检验 DNA 末端的 DNA-PKcs 自磷酸化对于末端连接是必要的这一假设。目标 3 将通过表征 DNA-PKcsKD/ 中的类别转换重组（由 A-EJ 和 NHEJ 介导）和 IgH-myc 致癌易位来测试 DNA-PKcs KD 蛋白物理阻断末端切除并抑制 A-EJ 和致癌易位的假设KD 细胞和小鼠。这些研究将共同​​确定 DNA-PKcs 磷酸化在 DNA 修复和易位中的作用。未来，DNA-PKcs磷酸化将为调节NHEJ和A-EJ癌症治疗提供一个有吸引力的靶点。