The Role of DNA Synthesis in Nonhomologous End Joining

描述（由申请人提供）：电离辐射杀死细胞，主要通过在染色体中引入双链断裂（DSB）引起基因组重排。这些染色体断裂通过同源重组或不太精确的非同源末端连接途径（NHEJ）修复。NHEJ也是V（D）J重组中有效修复DSB中间体的唯一途径。因此，NHEJ缺乏导致放射敏感性、严重免疫缺陷、遗传不稳定性和癌症易感性。1)NHEJ使用三种DNA聚合酶：pol lambda、pol mu和TdT。先前的工作表明，不同的底物要求对它们的生物作用有着至关重要的影响。我们现在将系统地比较这三种聚合酶在不同底物上的活性，以确定这三种聚合酶在同一修复途径中工作的优势。2)将研究三种相关但不同的聚合酶如何以及为什么与NHEJ特异性相关的结构基础。通过与两个结构生物学实验室的合作，我们将产生三种聚合酶的突变体和嵌合体。然后将使用复杂的无细胞和细胞测定法评估NHEJ期间这些变化对聚合酶活性的影响。3)同源重组虽然比NHEJ更精确，但通常对DNA合成的要求更高。然而，合成前体的水平在细胞周期和不同细胞类型中波动很大。我们将操纵合成前体的水平，并确定这是否是选择特定双链断裂修复途径的主要标准。NHEJ比大多数修复途径更不准确，但它仍然抑制肿瘤发生。这项研究将有助于调和这些观察结果：换句话说，我们将确定为什么NHEJ可能比替代方案更好。如我们的第三个目标所建议的，通过消耗DNA合成前体来操纵细胞修复途径的选择的能力也可以改善通过辐射的肿瘤治疗。通过非同源末端连接（NHEJ）修复染色体断裂的准确性低于大多数修复途径，但它仍然抑制肿瘤发生。这项研究将有助于调和这些观察结果：换句话说，我们将确定为什么NHEJ可能比替代方案更好。此外，如初步工作所建议的，操纵细胞修复途径的选择的简单能力可能导致通过辐射改善肿瘤的治疗。

DESCRIPTION (provided by applicant): Ionizing radiation kills cells and causes genome rearrangements primarily by introducing double strand breaks (DSBs) in chromosomes. These chromosome breaks are repaired by homologous recombination or the less accurate nonhomologous end joining pathway (NHEJ). NHEJ is also the only pathway for efficient repair of DSB intermediates in V(D)J recombination. Deficiency in NHEJ thus leads to radiosensitivity, severe immunodeficiency, genetic instability, and cancer predisposition. 1) NHEJ employs three DNA polymerases: pol lambda, pol mu, and TdT. Prior work hints that different substrate requirements have a critical impact on their biological role. We will now systematically compare activities of these three polymerases on different substrates, to determine what advantage there is to having all three work in the same repair pathway. 2) The structural basis for how and why three related, but different polymerases are specifically associated with NHEJ will be investigated. In collaboration with two structural biology labs we will generate mutant and chimeric versions of the three polymerases. The impact of these changes on polymerase activity during NHEJ will then be assessed using sophisticated cell-free and cellular assays. 3) Homologous recombination, though more accurate than NHEJ, is generally more demanding of DNA synthesis. However, levels of synthesis precursors fluctuate greatly during the cell cycle and in different cell types. We will manipulate levels of synthesis precursors, and determine if this is a major criteria for choosing a particular double strand break repair pathway. NHEJ is less accurate than most repair pathways, yet it still suppresses tumorigenesis. This research will help reconcile these observations: in other words, we will determine why NHEJ might be better than the alternative. The ability to manipulate the choice of cellular repair pathway by depletion of DNA synthesis precursors, as suggested in our third aim, could also improve treatment of tumors by radiation. Repair of chromosome breaks by nonhomologous end joining (NHEJ) is less accurate than most repair pathways, yet it still suppresses tumorigenesis. This research will help reconcile these observations: in other words, we will determine why NHEJ might be better than the alternative. Additionally, a facile ability to manipulate the choice of cellular repair pathway, as suggested by preliminary work, could lead to improved treatment of tumors by radiation.