Mechanism of Damage Tolerance by Nonhomologous End Joining

项目摘要/摘要 双链断裂(DSB)是在减数分裂、免疫系统中出现的致命dna损伤。 发展和癌症治疗。电离辐射(IR)和一些化疗药物通过诱导 DSB，它可能具有相关的端部损坏，从而阻止修复。修复受损的末端很重要 因为它决定了癌症治疗的效率和安全性。然而，之前的DSB修复研究已经 专注于未损坏的休息时间。拟议中的研究将确定受损末端修复的机制 通过非同源末端连接(NHEJ)，哺乳动物中主要的DSB修复途径。 NHEJ的最终步骤是通过DNA连接酶IV(Lig4)连接末端；我已经证明了它比 比其他连接受损末端的路径更有可能。Lig4仅在NHEJ复合体的上下文中起作用，因此它 目前还不清楚损伤耐受性是否是连接酶固有的。我假设Lig4是一种特殊的损坏 末端连接酶，这种活性对癌症治疗的反应是重要的。我正在创建Lig4的变体 单独的体外功能：它们有效地修复未受损的断裂，但不会损坏。我会产生细胞系 表达这些Lig4变异体，并测量受损DSB末端的修复。我会把这些细胞系暴露在 化疗药物和电离辐射以确定Lig4受损末端修复是否对 对癌症治疗的反应。我将确定NHEJ受损末端的容量是否与 细胞的放射敏感性和化学抗药性。这些结果将显示Lig4受损末端修复是否是 潜在的药物靶点，以改进现有的癌症治疗方法。 一些损坏的端部必须在修复之前进行处理。其中一个处理因素是DNA聚合酶Mu， 它填补了DSB末端的空白。令人惊讶的是，polu优先添加核糖核苷酸(RNA)，而不是 脱氧核糖核苷酸(DNA)，到DNA末端。初步数据显示，这些核苷酸刺激Lig4 活动，但尚不清楚这种情况发生的时间和原因。我推测波尔木添加的核苷酸 促进Lig4对受损末端的容错。我会确定什么时候核糖核苷酸对受损末端是重要的 修理。然后，我将产生Lig4的变种，取消核糖核苷酸的刺激。 我的结果将会揭示 这种将RNA引入基因组的惊人修复现象的生物学意义。 许多癌症疗法依赖于受损的双链断裂来特异性地杀死癌细胞。这个 拟议的研究将为人们对受损DSB修复过程知之甚少的过程提供机械性的见解。 我们还将确定损坏的DSB修复要求，这些要求可以有针对性地提高 癌症疗法。

Project Summary / Abstract Double strand breaks (DSBs) are lethal DNA lesions that arise during meiosis, immune system development, and cancer therapy. Ionizing radiation (IR) and some chemotherapeutics kill tumors by inducing DSBs, which can have associated end damage that blocks repair. Repair of damaged ends is important because it shapes the efficiency and safety of cancer therapy. However, previous DSB repair research has focused on undamaged breaks. The proposed research will determine the mechanisms of damaged end repair by nonhomologous end joining (NHEJ), the predominant DSB repair pathway in mammals. The ultimate step in NHEJ is the ligation of ends by DNA Ligase IV (Lig4); I have shown that it is more likely than other pathways to join damaged ends. Lig4 functions only in the context of the NHEJ complex so it is unclear whether damage tolerance is intrinsic to the ligase. I hypothesize that Lig4 is a specialized damaged end ligase, and this activity is important for the response to cancer therapy. I am creating variants of Lig4 that separate function in vitro: they efficiently repair undamaged, but not damaged breaks. I will generate cell lines expressing these Lig4 variants and measure repair of damaged DSB ends. I will expose these cell lines to chemotherapeutic agents and ionizing radiation to determine if Lig4 damaged end repair is important for the response to cancer therapy. I will determine whether NHEJ capacity on damaged ends corresponds to radiosensitivity and chemoresistance in cells. These results will show whether Lig4 damaged end repair is a potentially druggable target to improve existing cancer therapies. Some damaged ends must be processed prior to repair. One processing factor is DNA polymerase mu, which fills in gaps at DSB ends. Surprisingly, pol mu preferentially adds ribonucleotides (RNA), rather than deoxyribonucleotides (DNA), to DNA ends. Preliminary data suggests that these ribonucleotides stimulate Lig4 activity, but it is unclear when and why this happens. I hypothesize that ribonucleotides added by pol mu facilitate Lig4 tolerance of damaged ends. I will identify when ribonucleotides are important for damaged end repair. Then, I will generate variants of Lig4 that abolish its stimulation by ribonucleotides. My results will reveal the biological significance of this startling repair phenomenon which introduces RNA into the genome. Many cancer therapies rely on damaged double strand breaks to kill cancer cells with specificity. The proposed research will provide mechanistic insight into the poorly understood process of damaged DSB repair. We will also identify requirements for damaged DSB repair that can be targeted to improve the effectiveness of cancer therapies.