Non-catalytic functions of DNA-PK in DNA repair and tumorigenesis

DNA-PK 在 DNA 修复和肿瘤发生中的非催化功能

• 批准号: 8783374
• 负责人: Jennifer L Crowe
• 金额: $ 4.27万
• 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: 2014-09-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8783374
• 关键词: 1-Phosphatidylinositol 3-Kinase; Address; Affect; Antigen Receptors; Ataxia-Telangiectasia-Mutated protein kinase; B-Cell Development; B-Cell Lymphomas; B-Lymphocytes; Cancer Biology; Catalytic Domain; Cells; Chromosomal translocation; Code; DNA; DNA Double Strand Break; DNA Repair; DNA-PKcs; DNA-dependent protein kinase; Defect; Development; Double Strand Break Repair; Embryo; Embryonic Development; Excision; Family; Frequencies; Funding; G22P1 gene; Generations; Genomic Instability; Goals; Health; Human; IGH@ gene cluster; Immunodeficiency and Cancer; Immunoglobulin Class Switching; Immunoglobulin Genes; Immunoglobulin Switch Recombination; Immunoglobulins; KRP protein; Kinetics; Knock-in Mouse; Knockout Mice; Lead; Ligation; Lymphocyte; Lymphoid; Lymphoma; Lymphomagenesis; Malignant lymphoid neoplasm; Mediating; Modification; Mus; Mutation; Neurons; Nonhomologous DNA End Joining; Null Lymphocytes; Oncogenic; Pathway interactions; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological; Premalignant; Process; Proteins; Recurrence; Regulation; Reporter; Role; Sequence Homology; Severe Combined Immunodeficiency; System; Testing; Tumor Suppression; Tumor Suppressor Proteins; V(D)J Recombination; XRCC4 gene; XRCC5 gene; artemis; base; cell type; chemotherapy; cohort; congenital immunodeficiency; embryonic stem cell; member; neuron apoptosis; null mutation; prevent; progenitor; programs; protein structure; public health relevance; recombinational repair; repaired; seal; tumor; tumorigenesis

DESCRIPTION (provided by applicant): DNA repair is essential to maintaining cellular health and preventing tumorigenesis. This application seeks funding to study the normal function and regulation of the non-homologous end joining (NHEJ) factor DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and its substrate Artemis, during lymphocyte development and lymphomagenesis to elucidate how mutations in these proteins lead to severe combined immunodeficiency (SCID) and lymphomas. V(D)J recombination, the repair of programmed double-strand breaks (DSBs) in the Immunoglobulin (Ig) loci, is required for normal B cell development from lymphoid progenitors. DNA repair during V(D)J recombination requires the core NHEJ factors (Ku70, Ku80, Lig4, XRCC4, and XLF), and vertebrate specific DNA-PKcs and Artemis. While DNA-PKcs and Artemis are not strictly required for end-ligation, they are required for end processing such as opening hairpin-sealed coding ends during V(D)J recombination. Therefore, DNA-PKcs or Artemis null mutations result in isolated SCID, without the neuronal defects found with core-NHEJ deficiency. DNA-PKcs belongs to the PI3 Kinase related kinase family, which also includes ATM. ATM and DNA-PKcs share many common substrates, including DNA- PKcs itself. To determine whether DNA-PKcs protein has a role in DNA repair beyond its kinase activity, we knocked in the D3922A kinase dead (KD) mutation in murine DNA-PKcs (Prkdc). In contrast to the normal development of DNA-PK null mice, DNA-PKcsKD/KD, but not DNA-PKcs+/KD, mice died during late embryonic development with severe genomic instability, similar to core-NHEJ deficient mice (eg.Lig4). Surprisingly DNA- PKcsKD/KD mice were defective in end-ligation, but not in end processing (hairpin opening). Despite the severe genomic instability, DNA-PKcsKD/KDp53-/- mice have less lymphoma than the DNA-PKcs-/-p53-/- mice. Based on these and other findings, we hypothesize that the DNA-PKcs protein structure suppresses lymphomagenesis and supports normal lymphocyte development by regulating NHEJ, alternative-end- joining and oncogenic translocations. To test this, we propose to investigate (Aim 1) the noncatalytic function of DNA-PKcs in NHEJ and hairpin opening; (Aim 2) the noncatalytic function of DNA-PKcs in CSR and A-EJ; and (Aim 3) the role of DNA-PKcs in B cell lymphomagenesis. Together, these studies will determine the physiological function of the DNA-PKcs protein during DNA double strand break repair and lymphocyte development and how mutations in DNA-PKcs and its substrates leads to primary immunodeficiency and cancer.

描述（由申请人提供）：DNA修复对于维持细胞健康和预防肿瘤发生至关重要。该申请寻求资金，以研究正常功能和调节的非同源末端连接（NHEJ）因子DNA依赖性蛋白激酶催化亚基（DNA-PKcs），其底物Artemis，在淋巴细胞发育和淋巴瘤形成，以阐明如何在这些蛋白质突变导致严重的联合免疫缺陷（SCID）和淋巴瘤。V（D）J重组是免疫球蛋白（IG）基因座中程序性双链断裂（DSB）的修复，是淋巴祖细胞发育成正常B细胞所必需的。V（D）J重组过程中的DNA修复需要核心NHEJ因子（Ku 70、Ku 80、Lig 4、XRCC 4和XLF）以及脊椎动物特异性DNA-PKcs和Artemis。虽然DNA-PKcs和Artemis对于末端连接不是严格需要的，但它们对于末端加工是需要的，例如在V（D）J重组期间打开发夹密封的编码末端。因此，DNA-PKcs或Artemis无效突变导致孤立的SCID，而没有核心NHEJ缺陷所发现的神经元缺陷。DNA-PKcs属于PI 3激酶相关激酶家族，该家族还包括ATM。ATM和DNA-PKcs共享许多共同的底物，包括DNA-PKcs本身。为了确定DNA-PKcs蛋白是否在其激酶活性之外的DNA修复中具有作用，我们敲入小鼠DNA-PKcs（Prkdc）中的D3922 A激酶死亡（KD）突变。与DNA-PK缺失小鼠的正常发育相反，DNA-PKcs KD/KD，而不是DNA-PKcs+/KD，小鼠在胚胎发育晚期死亡，具有严重的基因组不稳定性，类似于核心-NHEJ缺陷小鼠（例如Lig 4）。令人惊讶的是，DNA-PKcsKD/KD小鼠在末端连接中有缺陷，但在末端加工（发夹打开）中没有缺陷。尽管存在严重的基因组不稳定性，但DNA-PKcs KD/KDp 53-/-小鼠的淋巴瘤比DNA-PKcs-/-p53-/-小鼠少。基于这些和其他研究结果，我们假设DNA-PKcs蛋白结构通过调节NHEJ、交替末端连接和致癌易位抑制淋巴瘤发生并支持正常淋巴细胞发育。为了验证这一点，我们建议研究（目的1）的非催化功能的DNA-PKcs在NHEJ和发夹打开;（目的2）的非催化功能的DNA-PKcs在CSR和A-EJ;和（目的3）的作用，DNA-PKcs在B细胞淋巴瘤发生。总之，这些研究将确定DNA-PKcs蛋白在DNA双链断裂修复和淋巴细胞发育过程中的生理功能，以及DNA-PKcs及其底物的突变如何导致原发性免疫缺陷和癌症。