Roles of Telomeric Oxidative DNA Lesions in Telomere Length Regulation

该项目的目标是确定氧化DNA损伤调节端粒的机制。 DNA结构、获得端粒蛋白、端粒酶活性和端粒长度的动态平衡。 氧化还原调节功能障碍在癌症中很常见，并会增加活性氧物种，从而 产生致突变和细胞毒性的DNA损伤。端粒的维持对基因组的稳定是必不可少的，但 它们极易受到氧化损伤。大多数癌症通过上调端粒来防止端粒侵蚀 端粒酶。这种酶延长了端粒的单链突起，端粒可以自我折叠成稳定的 二次结构。我们将测试氧化DNA损伤增加动态弹性的假说 端粒突出，从而改变了可及性和端粒酶活性。为此，我们开发了一个单一的 测量结构动力学的分子荧光共振能量转移检测系统 悬垂构象和端粒酶延伸活性的实时检测。使用这一创新方法，我们 发现了几个令人惊讶的、以前无法达到的结果。我们的初步研究表明，单一的8- 氧鸟嘌呤损伤增加了端粒蛋白POT1的悬垂动力学，并加速了端粒蛋白POT1的负载。 我们的生化研究一直表明，8-氧鸟嘌呤能诱导强大的端粒酶活性和 在其他情况下无法接近的悬挑上的处理能力。目标1将定义氧化损伤如何影响 端粒突出体现了结构的多样性、动力学和可及性。我们会用8-氧鸟嘌呤测试悬垂 在不同的位置，以及最常见的氧化胸腺嘧啶损伤，胸腺嘧啶二醇，它施加了一个 对复制的强大阻止。目标2将研究氧化损伤如何调节端粒酶可及性和 活动。有DNA损伤的突出物上的端粒酶活性将在没有和存在的情况下进行检测 端粒酶活性因子POT1-TPP1。对于目标1和目标2，补充生化实验将是 执行以验证和解释单分子结果。目标3将研究氧化损伤是如何 调节端粒长度和端粒酶在人类细胞中的募集。一般氧化应激 将在促氧化剂条件下诱导，而8-氧鸟嘌呤在端粒上的诱导将通过 一种创新的氟激活多肽靶向系统。荧光原位杂交将用于 测量端粒长度并定位细胞中的端粒酶。我们将检测端粒长度和端粒酶 在缺乏修复特定DNA损伤所需的不同糖基酶的细胞中进行募集。这些研究 将为DNA氧化损伤如何影响染色体末端的处理提供重要的见解 结构。这个项目将填补我们在理解普遍的氧化应激和8- 特别是氧鸟嘌呤，改变了端粒的维持。最终，这些知识将对 开发新的策略：1)保存端粒以减轻氧化应激对健康细胞的影响 或者相反，这2)抑制恶性细胞中的端粒酶以阻止增殖。

The goals of this project are to define the mechanisms by which oxidative DNA damage regulates telomeric DNA structure, access to telomeric proteins, telomerase activity and telomere length homeostasis. Dysfunctional redox regulation is common among cancers and elevates reactive oxygen species, which generate mutagenic and cytotoxic DNA lesions. Telomere maintenance is essential for genome stability, yet they are highly susceptible to oxidative damage. Most cancers prevent telomere erosion by upregulating telomerase. This enzyme extends the telomeric single-stranded overhang, which can self-fold into stable secondary structures. We will test the hypothesis that oxidative DNA lesions increase the dynamic flexibility in the telomeric overhang thereby altering accessibility and telomerase activity. For this, we developed a single molecule fluorescence resonance energy transfer detection system that measures structural dynamics in overhang conformation and telomerase extension activity in real time. Using this innovative approach we uncovered several surprising and previously unattainable results. Our preliminary studies show that a single 8- oxoguanine lesion increases the overhang dynamics and accelerates loading of telomeric protein POT1. Consistently, our biochemical studies reveal that an 8-oxoguanine induces robust telomerase activity and processivity on overhangs that are otherwise inaccessible. Aim 1 will define how oxidative lesions impact the telomeric overhang structural diversity, dynamics and accessibility. We will test overhangs with 8-oxoguanine at various positions, along with the most common oxidized thymine lesion, thymine glycol, which imposes a strong block to replication. Aim 2 will examine how oxidative lesions modulate telomerase accessibility and activity. Telomerase activity on overhangs with DNA lesions will be tested in the absence and presence of telomerase processivity factor POT1-TPP1. For aims 1 and 2, complementary biochemical experiments will be performed to validate and interpret the single molecule results. Aim 3 will examine how oxidative lesions modulate telomere length and telomerase recruitment to telomeres in human cells. General oxidative stress will be induced with pro-oxidant conditions, whereas 8-oxoguanine induction at telomeres will be achieved by an innovative fluorogen activated peptide targeting system. Fluorescent in situ hybridization will be used to measure telomere length and to localize telomerase in cells. We will examine telomere length and telomerase recruitment in cells lacking distinct glycosylases that are required to repair specific DNA lesions. These studies will provide crucial insights into how oxidative DNA damage impacts the processing of chromosome end structures. This project will fill a significant void in our understanding of how general oxidative stress and 8- oxoguanine, in particular, alters telomere maintenance. Ultimately, this knowledge will be highly valuable for developing new strategies that 1) preserve telomeres to mitigate the effects of oxidative stress on healthy cells or conversely, that 2) inhibit telomerase in malignant cells to halt proliferation.