Mechanisms of Telomere Resistance to DNA Lesion Removal

描述（由申请人提供）：该项目的目标是确定端粒核苷酸表达修复（NER）抑制的机制和程度，以及未修复的 DNA 损伤对端粒结构和功能的影响。染色体末端的端粒对于基因组稳定性和持续细胞增殖至关重要。端粒 DNA 非常容易受到紫外线 (UV) 光引起的光产物形成的影响，而这些光产物会被大量基因组中的 NER 去除。该提案将检验端粒结合蛋白通过抑制 NER 酶的酶活性来防止端粒 DNA 损伤去除的假设。我们的初步研究表明，光产物通过干扰端粒复制而导致端粒丢失和畸变，这与端粒病变去除的缺陷一致。我们进一步表明，端粒蛋白在体外抑制 NER 所需的核酸酶的催化​​活性。目标 1 将比较 UVC 照射的 NER 充足细胞和缺陷细胞的端粒损伤和功能障碍的终点，以确定光产物如何影响个体端粒。我们将使用一种创新的测定方法来测量端粒中的光产物修复率，与大量基因组相比，该方法可以量化从 UVC 暴露的人体细胞中分离出的端粒中的光产物。目标 2 将使用荧光蛋白标签和活细胞成像来测试细胞核中关键 NER 蛋白与非端粒区域的招募情况，以招募到受损的端粒区域。激光微照射将用于在细胞核的特定区域产生光产物和大体积加合物。目标 3 将研究端粒蛋白如何调节 NER 过程中的各种酶促步骤。将在存在单个端粒蛋白或完整端粒蛋白复合物的情况下，使用特定端粒和非端粒基质上的细胞提取物在体外检查 NER。 NER 是去除由环境遗传毒物和抗癌药物产生的各种 DNA 损伤所必需的。该项目将填补我们的一个重大空白 了解端粒如何逃避 NER，以及未修复的 DNA 损伤如何改变端粒结构和功能。这些知识对于制定新策略非常有价值，这些策略 1）保护端粒以减轻环境基因毒物暴露的影响，或相反，2）抑制全局基因组 NER 以使恶性细胞对抗癌基因毒药的杀伤敏感。

DESCRIPTION (provided by applicant): The goals of this project are to define the mechanisms and extent of nucleotide expression repair (NER) inhibition at telomeres, and the impact of unrepaired DNA lesions on telomere structure and function. Telomeres at chromosome ends are essential for genome stability and sustained cell proliferation. Telomeric DNA is highly susceptible to photoproduct formation caused by ultraviolet (UV) light, which are removed by NER in the bulk genome. This proposal will test the hypothesis that telomere binding proteins prevent DNA lesion removal at telomeres by inhibiting the enzymatic activities of NER enzymes. Our preliminary studies show that photoproducts induce telomere loss and aberrations by interfering with telomere replication, which is consistent with a deficiency in lesion removal at telomeres. We further show that a telomeric protein inhibits the catalytic activity of a nuclease required for NER in vitro. Aim 1 will compare endpoints of telomeric damage and dysfunction in UVC irradiated NER proficient- and deficient- cells to establish how photoproducts impact individual telomeres. We will measure photoproduct repair rates in telomeres, compared to the bulk genome, using an innovative assay that quantifies photoproducts in telomeres isolated from UVC exposed human cells. Aim 2 will test for recruitment of key NER proteins to damaged telomeric regions, compared with non-telomeric regions, in cell nuclei using fluorescent protein tags and live cell imaging. Laser micro-irradiatin will be used to generate photoproducts and bulky adducts at define regions in the cell nucleus. Aim 3 will examine how telomeric proteins modulate various enzymatic steps in the NER process. NER will be examined in vitro using cell extracts on defined telomeric and non-telomeric substrates in the presence of individual telomeric proteins or the complete telomeric protein complex. NER is required for removing a wide variety of DNA lesions generated by environmental genotoxicants and anti-cancer drugs. This project will fill a significant void in our understanding of how telomeres evade NER, and how unrepaired DNA lesions alter telomere structure and function. This knowledge will be highly valuable for developing new strategies that 1) preserve telomeres to mitigate the effects of environmental genotoxicant exposures or conversely, that 2) inhibit global genome NER to sensitize malignant cells for killing by anti-cancer genotoxic drugs.