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Mechanisms of Telomere Resistance to DNA Lesion Removal

端粒对 DNA 损伤去除的抵抗机制

基本信息

批准号：
9277466
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
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-01 至 2019-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9277466
关键词：
Affect Aging 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 Dangerousness 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 Recruitment Activity Resistance Role Skin Aging Skin Cancer Structure Telomere-Binding Proteins Testing Ultraviolet Rays adduct cancer cell carcinogenesis cell growth cell killing chromatin immunoprecipitation environmental agent environmental mutagens enzyme activity experience genotoxicity in vitro activity innovation irradiation killings live cell imaging member novel therapeutic intervention nuclease overexpression prevent protein complex public health relevance repair enzyme repaired telomere telomere loss tumor ultraviolet ultraviolet irradiation

项目摘要

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）：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于基因组稳定性和持续的细胞增殖是必不可少的。端粒DNA对由紫外线（UV）光引起的光产物形成高度敏感，所述光产物在本体基因组中被NER去除。这项建议将测试的假设，端粒结合蛋白阻止DNA损伤去除端粒通过抑制NER酶的酶活性。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，这与端粒损伤去除的缺陷一致。我们进一步表明，端粒蛋白抑制体外NER所需的核酸酶的催化活性。目的1将比较UVC照射NER熟练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，该方法定量从UVC暴露的人类细胞中分离的端粒中的光产物。目标2将测试募集的关键NER蛋白质损伤的端粒区域，与非端粒区域相比，在细胞核中使用荧光蛋白标签和活细胞成像。激光微照射将用于在细胞核中的限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。在存在单个端粒蛋白或完整端粒蛋白复合物的情况下，使用细胞提取物对规定的端粒和非端粒底物进行体外检查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。这个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损伤如何改变端粒的结构和功能。这些知识对于开发新的策略将是非常有价值的，这些策略1）保护端粒以减轻环境遗传毒性暴露的影响，或者相反，2）抑制全局基因组NER以使恶性细胞敏感，从而被抗癌遗传毒性药物杀死。