Mechanisms of Telomere Resistance to DNA Lesion Removal

端粒对 DNA 损伤去除的抵抗机制

• 批准号: 9064774
• 负责人: Patricia L Opresko
• 金额: $ 32.12万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9064774
• 关键词: Affect; Antineoplastic Agents; Apoptosis; Binding; Biological Assay; Biological Preservation; Biology; Cell Extracts; Cell Nucleus; Cell Proliferation; Cell Survival; Cells; Chromosome abnormality; Chromosomes; Cleaved cell; Collaborations; Complex; DNA; DNA Damage; DNA Repair; DNA Repair Pathway; DNA Structure; DNA lesion; DNA photoproducts; Data; Defect; Degenerative Disorder; Development; ERCC1 gene; Enzymes; Excision; Functional disorder; Genome; Genome Stability; Genomic Instability; Genomics; Genotoxic Stress; Goals; Health; Human; In Vitro; Individual; Knowledge; Lasers; Lesion; Malignant Neoplasms; Measures; Normal Cell; Nucleic Acids; Nucleotide Excision Repair; Nucleotides; Pathway interactions; Pharmaceutical Preparations; Positioning Attribute; Process; Proteins; Resistance; Role; Skin Aging; Skin Cancer; Structure; Telomere-Binding Proteins; Testing; Ultraviolet Rays; adduct; age related; cancer cell; carcinogenesis; cell growth; cell killing; chromatin immunoprecipitation; environmental agent; environmental mutagens; enzyme activity; experience; in vitro activity; innovation; irradiation; killings; live cell imaging; member; novel therapeutic intervention; nuclease; prevent; protein complex; repair enzyme; repaired; telomere; telomere loss; tumor; ultraviolet; ultraviolet irradiation

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.

描述（由申请人提供）：该项目的目标是定义端粒处核苷酸表达修复（NER）抑制的机制和程度，以及未修复的DNA病变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非常容易受到由紫外线（UV）光引起的光产物形成，这些光产光被NER在大体基因组中去除。该建议将检验以下假设：端粒结合蛋白通过抑制NER酶的酶促活性来防止端粒中DNA病变的去除。我们的初步研究表明，光产物通过干扰端粒复制而诱导端粒损失和畸变，这与端粒中病变的缺乏一致。我们进一步表明，端粒蛋白抑制了体外所需的核酸酶的催化​​活性。 AIM 1将比较UVC辐照的端粒损伤和功能障碍的终点，以确定光产物如何影响单个端粒的细胞。我们将使用一种创新的测定法对端粒的光产物修复率进行测量，该测定法量化了从UVC暴露的人类细胞中分离出的端粒中的光产物。 AIM 2将使用荧光蛋白标签和活细胞成像在细胞核中测试与非层次区域相比，在细胞核中募集关键NER蛋白为受损的端粒区域。激光微iRradiatin将用于在细胞核中定义区域产生光产物和笨重的加合物。 AIM 3将检查端粒蛋白如何在NER过程中调节各种酶促步骤。在存在单个端粒蛋白或完整的端粒蛋白复合物的情况下，将使用在定义的端粒和非telomeric底物上使用细胞提取物在体外检查NER。消除环境遗传毒性和抗癌药物产生的多种DNA病变所必需的NER。这个项目将在我们的 了解端粒如何逃避NER以及未修复的DNA病变如何改变端粒结构和功能。对于制定新策略，该知识将非常有价值1）保留端粒以减轻环境基因毒性暴露的作用，或者相反，这将是2）抑制全球基因组NER，以使恶性细胞通过抗癌基因毒性药物杀死恶性细胞。