Roles of Telomeric Oxidative DNA Lesions in Telomere Length Regulation

本项目的目标是确定DNA氧化损伤调节端粒的机制， DNA结构、端粒蛋白的获取、端粒酶活性和端粒长度稳态。 功能失调的氧化还原调节在癌症中很常见，并升高活性氧， 产生致突变和细胞毒性DNA损伤。端粒的维持对基因组的稳定性至关重要， 它们对氧化损伤高度敏感。大多数癌症通过上调 端粒酶这种酶延伸端粒单链突出端，其可以自我折叠成稳定的 二级结构。我们将检验氧化性DNA损伤增加细胞内的动态灵活性的假设。 端粒突出端从而改变可接近性和端粒酶活性。为此，我们开发了一个 分子荧光共振能量转移检测系统，其测量 真实的时间的突出端构象和端粒酶延伸活性。通过这种创新的方法，我们 发现了一些令人惊讶的和以前无法实现的结果。我们的初步研究表明，一个8- 氧代鸟嘌呤损伤增加了突出动力学并加速端粒蛋白POT 1的加载。 因此，我们的生物化学研究表明，8-氧代鸟嘌呤诱导强大的端粒酶活性， 在其他方式无法到达的突出物上的持续合成能力。目标1将定义氧化损伤如何影响 端粒突出端结构多样性、动态和可及性。我们将用8-氧代鸟嘌呤测试突出端 在不同的位置，沿着最常见的氧化胸腺嘧啶损伤，胸腺嘧啶乙二醇，这将施加一个 强大的复制块。目的2将研究氧化损伤如何调节端粒酶的可及性， 活动将在不存在和存在以下物质的情况下测试具有DNA损伤的突出端上的端粒酶活性： 端粒酶合成因子POT 1-TPP 1。就目标1和2而言，将提供补充性生化实验， 用于验证和解释单分子结果。目标3将研究氧化损伤如何 调节人类细胞中端粒长度和端粒酶向端粒的募集。一般氧化应激 将用促氧化剂条件诱导，而端粒处的8-氧代鸟嘌呤诱导将通过 一个创新的荧光激活肽靶向系统。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化酶的细胞中进行募集。这些研究 将为氧化性DNA损伤如何影响染色体末端的加工提供重要的见解 结构.该项目将填补我们对一般氧化应激和8-羟色胺的理解中的一个重要空白。 特别是氧代鸟嘌呤改变端粒的维持。最终，这些知识将非常有价值， 开发新的策略，1）保护端粒，以减轻氧化应激对健康细胞的影响 或相反，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.