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

描述(由申请人提供)：DNA修复是维持细胞健康和防止肿瘤发生的关键。这项申请寻求资金，以研究非同源末端连接(NHEJ)因子DNA依赖的蛋白激酶催化亚单位(DNA-PKcs)及其底物Artemis在淋巴细胞发育和淋巴肿大过程中的正常功能和调节，以阐明这些蛋白的突变如何导致严重的联合免疫缺陷(SCID)和淋巴瘤。V(D)J重组，即免疫球蛋白(Ig)基因座上程序性双链断裂(DSB)的修复，是从淋巴祖细胞正常发育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(Ku70、Ku80、Lig4、XRCC4和XLF)以及脊椎动物特有的DNA-PKcs和Artemis。虽然DNA-PKcs和Artemis不是末端连接所必需的，但它们是末端加工所必需的，例如在V(D)J重组过程中打开发夹密封的编码端。因此，DNA-PKcs或Artemis零突变会导致孤立的SCID，而不会出现core-NHEJ缺陷所导致的神经元缺陷。DNA-PKcs属于PI3激酶相关蛋白家族，也包括ATM。ATM和DNA-PKcs有许多共同的底物，包括DNA-PKcs本身。为了确定DNA-PKcs蛋白是否在其激酶活性以外的DNA修复中发挥作用，我们在小鼠DNA-PKcs(Prkdc)中敲入了D3922A激酶死亡(KD)突变。与DNA-PK缺失小鼠(DNA-PKcsKD/KD，而不是DNA-PKcs+/KD)的正常发育相反，小鼠在胚胎发育后期死亡，基因组严重不稳定，类似于core-NHEJ缺陷小鼠(如图4)。令人惊讶的是，DNA-PKcsKD/KD小鼠在末端连接方面存在缺陷，但在末端加工(发夹打开)方面没有缺陷。尽管存在严重的基因组不稳定性，DNA-PKcsKD/KDp53-/-小鼠的淋巴瘤发病率低于DNA-PKcs-/-P53-/-小鼠。基于这些和其他发现，我们假设DNA-PKcs蛋白结构通过调节NHEJ、选择性末端连接和致癌易位来抑制淋巴肿大并支持正常的淋巴细胞发育。为了验证这一点，我们建议研究(目的1)DNA-PKcs在NHEJ和发夹开放中的非催化作用；(目的2)DNA-PKcs在CSR和A-EJ中的非催化作用；(目的3)DNA-PKcs在B细胞淋巴瘤发生中的作用。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免疫缺陷和癌症。

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.