Oxidative DNA base damage and repair at telomeres and the relevance to cell senes

端粒氧化 DNA 碱基损伤和修复及其与细胞功能的相关性

• 批准号: 8566953
• 负责人: Li Lan
• 金额: $ 19.06万
• 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: 2013-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8566953
• 关键词: Address; Affect; Age; Aging; Antibodies; Apoptosis; Base Excision Repairs; Binding; Binding Sites; Biological Preservation; Cell Aging; Cell Death; Cell Nucleus; Cell Survival; Cell division; Cells; Chromatin; Chromosomes; Complex; Confocal Microscopy; DNA; DNA Damage; DNA Repair; Data; Disease; Engineering; Functional disorder; Future; Genome; Genomics; Goals; Guanine; Human; In Vitro; Kinetics; Lead; Life; Light; Measures; Methods; Microscopy; Outcome; Oxidative Stress; Pathology; Pathway interactions; Photosensitizing Agents; Population; Positioning Attribute; Production; Protein Binding; Proteins; Reactive Oxygen Species; Reporting; Repressor Proteins; Resolution; Site; Structure; System; TERF1 gene; Telomere Maintenance; Telomere Shortening; Telomere-Binding Proteins; Tertiary Protein Structure; Testing; Tetanus Helper Peptide; Time; Work; age related; base; cell age; cell type; experience; healthy aging; innovation; insight; irradiation; oxidative damage; prevent; public health relevance; repaired; research study; response; senescence; telomere

DESCRIPTION (provided by applicant): Telomeres at chromosome ends shorten during cell division and aging in humans. Critically short dysfunctional telomeres trigger cell senescence or apoptosis, which contributes to aging-related diseases and degeneration. Oxidative stress accelerates telomere shortening, and generates reactive oxygen species (ROS), which are particularly damaging to telomeric TTAGGG repeat sequences. The goals of this proposal are to 1) measure the response of base excision repair (BER) proteins to DNA damage at telomeric regions compared with non- telomere regions; and 2) to define the impact of oxidized telomeric DNA on telomere integrity and telomere functions in preventing cell senescence and cell death. A critical barrier to investigating DNA damage and repair at telomeres has been an inability to target damage to the telomeres. To overcome this obstacle we developed a highly innovative system for confining ROS-induced DNA damage to defined regions in the genome. For this we fused KillerRed (KR) protein, a photosensitizer that generates ROS upon light irradiation, to a Tet-repressor (tetR) protein that binds to a single engineered site within condensed chromatin. We established that oxidative damage occurred only at the tetR bound site. To target telomeres we fused KR to the telomere binding protein TRF1 and showed that this restricts ROS-induced damage to the telomeres. In Aim 1, we will combine the KR system with confocal microscopy to visualize the real time damage response of BER proteins to targeted oxidative damage in telomeric and non-telomeric genomic regions, with 3D resolution in a single cell nucleus. We will measure the mobilization kinetics of various GFP-tagged BER proteins, and the protein domains required for the response, to ROS damage at the TRF1 bound telomeric sites compared with the tetR bound non-telomeric site. In Aim 2, we will test several endpoints of telomere damage and dysfunction, including cellular senescence and apoptosis, after ROS production by activating KR with light exposure in cells that stably express KR-TRF1, compared with cells expressing HcRed-TRF1 as a control. This study will include both bulk cell population and single cell experiments. Using the microscopy system to target KR activation to defined numbers of KR-TRF1 foci, we will measure the average number of "oxidized" telomeres required to trigger apoptosis or senescence. Successful completion of the project will provide crucial insights into how oxidative stress accelerates telomere shortening, and how telomeric oxidative base damage impacts telomere function and promotes cell senescence. The results will pave the way for future work examining how telomeric damage and repair change with age and vary with cell type, and should lead to new strategies for preserving telomere function to promote healthy aging. !

描述（由申请人提供）：染色体末端的端粒在人类细胞分裂和衰老过程中缩短。极短的功能失调端粒会引发细胞衰老或凋亡，从而导致与衰老相关的疾病和退化。氧化应激加速端粒缩短，并产生活性氧 (ROS)，这对端粒 TTAGGG 重复序列尤其有害。该提案的目标是 1) 测量端粒区域与非端粒区域相比碱基切除修复 (BER) 蛋白对 DNA 损伤的反应； 2) 确定氧化端粒 DNA 对端粒完整性和端粒预防细胞衰老和细胞死亡功能的影响。研究端粒 DNA 损伤和修复的一个关键障碍是无法针对端粒损伤。为了克服这一障碍，我们开发了一种高度创新的系统，用于将 ROS 诱导的 DNA 损伤限制在基因组中的特定区域。为此，我们将 KillerRed (KR) 蛋白（一种在光照射下产生 ROS 的光敏剂）与 Tet 阻遏物 (tetR) 蛋白融合，后者与浓缩染色质内的单个工程位点结合。我们确定氧化损伤仅发生在 tetR 结合位点。为了靶向端粒，我们将 KR 与端粒结合蛋白 TRF1 融合，并表明这限制了 ROS 诱导的端粒损伤。在目标 1 中，我们将 KR 系统与共聚焦显微镜相结合，以单细胞核中的 3D 分辨率可视化 BER 蛋白对端粒和非端粒基因组区域中的靶向氧化损伤的实时损伤反应。我们将测量各种 GFP 标记的 BER 蛋白的动员动力学，以及响应 TRF1 结合端粒位点与 tetR 结合非端粒位点处 ROS 损伤所需的蛋白结构域。在目标 2 中，我们将在稳定表达 KR-TRF1 的细胞中，与表达 HcRed-TRF1 的细胞作为对照，通过光照激活 KR，测试 ROS 产生后端粒损伤和功能障碍的几个终点，包括细胞衰老和凋亡。这项研究将包括大量细胞群和单细胞实验。使用显微镜系统将 KR 激活靶向指定数量的 KR-TRF1 焦点，我们将测量触发细胞凋亡或衰老所需的“氧化”端粒的平均数量。该项目的成功完成将为氧化应激如何加速端粒缩短，以及端粒氧化碱基损伤如何影响端粒功能并促进细胞衰老提供重要见解。这些结果将为未来研究端粒损伤和修复如何随年龄变化以及随细胞类型变化而变化的工作铺平道路，并应催生保护端粒功能以促进健康衰老的新策略。 ！