DNA-PKCS Phosphorylation in DNA Repair and Chromosomal Translocations

描述（由申请方提供）：涉及IG或TCR基因座的复发性致癌性易位是人类淋巴恶性肿瘤的特征。这些易位通常来源于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末端的获得、正确的末端加工和末端错接是染色体易位的三个基本步骤。淋巴细胞中发育的DSB通常通过非同源末端连接（NHEJ）途径在交替末端连接（A-EJ）途径的帮助下修复，所述交替末端连接（A-EJ）途径优先在连接处使用微同源性（MH）。NHEJ还负责末端加工，例如在V（D）重组期间产生的IG/ TCR基因座处打开发夹末端。序列分析表明，NHEJ和A-EJ也介导了产生易位的“错误”修复。因此，了解NHEJ和A-EJ的机制和调节在淋巴瘤发生中具有广泛的意义。DNA-PKcs是DNA依赖性蛋白激酶（DNA-PK）的催化亚基，是NHEJ因子和PI 3 K相关蛋白激酶。在没有DNA-PKcs的情况下，直接末端连接在很大程度上是正常的，但发夹开口-连接前密封发夹末端所需的末端加工形式，被完全阻断。在这里，我们报告，在一个敲入小鼠模型表达激酶死亡（KD）形式的DNA-PKcs单独（DNA-PKcs KD/KD），直接末端连接被完全阻断，导致胚胎致死率类似于核心NHEJ缺陷（如Lig 4-/-）小鼠。Ku 70的共缺失是DNA-PKcs募集到DNA所必需的，拯救了DNA-PKcs KD/KD小鼠的胚胎发育，表明DNA-PKcs蛋白调节DNA末端的末端连接。尽管末端连接缺陷，DNA-PKcsKD/KD细胞正常打开发夹，但仅在正常ATM激酶活性存在下，揭示了ATM在发夹打开中的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反，尽管存在严重的基因组不稳定性，但DNA-PKcsKD/KDp 53-/-小鼠中的淋巴瘤罕见，表明A-EJ中存在潜在缺陷。DNA-PKcs自身磷酸化，并在DSB后被ATM磷酸化。基于这些发现，我们假设DNA-PKcs磷酸化调节发夹开放（Aim 1），末端连接（Aim 2）和A-EJ（Aim 3），以抑制淋巴瘤的发生。Aim 1将解决如何通过ATM和DNA-PKcs促进发夹开放的DNA-PKcs和Artemis的冗余磷酸化。目的2通过鉴定DNA-PKcs上功能相关的自磷酸化位点，验证DNA-PKcs在DNA末端的自磷酸化是末端连接所必需的假说。目的3将通过表征DNA-PKcs KD/KD细胞和小鼠中的类别转换重组（由A-EJ和NHEJ介导）和IgH-myc致癌易位来检验DNA-PKcs KD蛋白物理地阻断末端切除并抑制A-EJ和致癌易位的假设。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。在未来，DNA-PKcs磷酸化将提供一个有吸引力的目标来调节NHEJ和A-EJ用于癌症治疗。

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.