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

描述(申请人提供)：人类染色体末端的端粒在细胞分裂和衰老过程中变短。功能异常的端粒过短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，并产生活性氧物种(ROS)，这对端粒TTAGGG重复序列尤其有害。本研究的目的是1)比较端粒和非端粒区域的碱基切除修复(BER)蛋白对DNA损伤的反应；2)确定氧化的端粒DNA对端粒完整性和端粒功能的影响，以防止细胞衰老和细胞死亡。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进行定位。为了克服这一障碍，我们开发了一种高度创新的系统，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(一种在光照射时产生ROS的光敏剂)与Tet-repressor(TetR)蛋白融合在一起，TetR蛋白与浓缩染色质中的单个工程位点结合。我们证实氧化损伤只发生在TetR结合部位。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对端粒的损伤。在目标1中，我们将结合KR系统和共聚焦显微镜来可视化BER蛋白对端粒和非端粒基因组区域靶向氧化损伤的实时损伤响应，并在单个细胞核中进行3D分辨率分析。我们将测量各种GFP标记的BER蛋白在TRF1结合的端粒位点和TetR结合的非端粒位点对ROS损伤的动员动力学以及反应所需的蛋白结构域。在目标2中，我们将通过在稳定表达KR-TRF1的细胞中通过光照激活KR来测试端粒损伤和功能障碍的几个终点，包括细胞衰老和凋亡，并与表达HcRed-TRF1的细胞作为对照。这项研究将包括大量细胞群体和单细胞实验。使用显微镜系统将KR激活定位于指定数量的KR-TRF1焦点，我们将测量触发细胞凋亡或衰老所需的平均“氧化”端粒数量。该项目的成功完成将为了解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和促进细胞衰老提供重要的见解。这一结果将为未来研究端粒损伤和修复如何随着年龄和细胞类型的变化而变化铺平道路，并将导致保护端粒功能以促进健康衰老的新策略。好了！

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. !