Mechanisms of Telomere Resistance to DNA Lesion Removal

描述（由申请人提供）：本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基因组的稳定和细胞的持续增殖至关重要。端粒DNA对紫外线（UV）引起的光产物形成非常敏感，这些光产物在基因组中被NER去除。这一提议将验证端粒结合蛋白通过抑制NER酶的酶活性来阻止端粒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诱导端粒丢失和畸变，这与端粒病变去除的缺陷是一致的。我们进一步表明，在体外，端粒蛋白抑制了NER所需的核酸酶的催化活性。目的1将比较UVC照射下的NER精通和缺陷细胞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。我们将测量端粒中的光产物修复率，与大量基因组相比，使用一种创新的测定方法，定量从暴露于UVC的人类细胞中分离的端粒中的光产物。目的2将使用荧光蛋白标签和活细胞成像技术，检测细胞核中受损端粒区与非端粒区相比，关键NER蛋白的募集情况。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。目的3将研究端粒蛋白如何调节NER过程中的各种酶促步骤。将在体外使用细胞提取物在特定的端粒和非端粒底物上检测NER，在存在单个端粒蛋白或完整的端粒蛋白复合物的情况下。NER是清除由环境基因毒物和抗癌药物产生的多种DNA损伤所必需的。这个项目将填补我们的一个重大空白

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.