Scanning Correlation Microscopy Methods for Quantifying DNA Repair Kinetics

用于量化 DNA 修复动力学的扫描相关显微镜方法

• 批准号: 8031079
• 负责人: Georgios Alexandrakis
• 金额: $ 16.71万
• 依托单位: UNIVERSITY OF TEXAS ARLINGTON
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8031079
• 关键词: Binding; Bleomycin; Catalytic Domain; Cell Nucleus; Cells; Characteristics; Chemical Agents; DNA; DNA Damage; DNA Repair; DNA repair protein; DNA-dependent protein kinase; Data; Diffusion; Dose; Exposure to; Fiber; Fluorescence; Fluorescence Microscopy; Fluorescence Recovery After Photobleaching; Gene Mutation; Generations; Hydrogen Peroxide; Image; Ionizing radiation; Kinetics; Lasers; Life; Lighting; Masks; Measures; Methodology; Methods; Microscopy; Modeling; Molecular; Monitor; Nature; Pattern; Physiologic pulse; Proteins; Protocols documentation; Quantitative Microscopy; Research Personnel; Resistance; Roentgen Rays; Scanning; Signal Transduction; Site; Spectrum Analysis; Spottings; System; Techniques; Testing; Time; Variant; Work; cancer cell; cancer therapy; design; established cell line; improved; in vivo; novel; photoactivation; protein aggregation; repaired; research study; resistance mechanism; simulation; two-photon

DESCRIPTION (provided by applicant): The kinetics of most fluorescently tagged DNA repair proteins subsequent to exposure to ionizing radiation, or radiomimetic chemical agents, cannot be quantified in the living cell without serious perturbation of the system under study. With the exception of only few proteins that attach in large numbers near DNA damage sites (e.g. 3-H2AX, 53BP1), most other proteins attach in fewer copies near the DNA damage sites and cannot be visualized by fluorescence microscopy. This because of the high background from freely moving, or immobile, fluorescent proteins that mask the weak aggregation of DNA repair proteins at damage sites. As a result, DNA damage is usually visualized by inducing clustered DNA damage by illumination with a laser beam. This illumination induces very high accumulation of repair proteins in one or more large spots in the nucleus. Nevertheless, the laser-induced damage is complicated in nature and may not be a good surrogate to ionizing radiation or radiomimetic agents for studying DNA repair in vivo. In this work we propose to develop quantitative microscopy methods that can overcome the current major limitation of not being able to quantify the kinetics of fluorescently tagged DNA repair proteins at sparse DNA damage sites. More specifically we will apply raster image correlation spectroscopy (RICS), a technique that analyzes the spatio-temporal fluorescence intensity fluctuations in image pixels, to quantify the repair kinetics of proteins with sparse accumulation in the cell nucleus. We will use RICS to quantify the repair kinetics of the DNA-dependent protein kinase catalytic subunit (DNA-PKCS), in its wild type and repair-deficient 7A forms after exposure to 3-rays and bleomycin that are both double strand break forming agents. We will also test hydrogen peroxide, a single strand break (DSB) forming agent, as a negative control. We will show that the DNA-PKCS kinetics after formation of DSBs can be quantified by RICS. Furthermore we propose to develop two specialized forms of RICS to further enhance the quantification of DNA repair kinetics of these proteins: (i) Photo-Activation RICS (PA-RICS) will offer control of the fluorescently tagged repair protein concentration, and (ii) Coherent Control RICS (CC-RICS) will optimize laser pulse characteristics to enhance fluorescence emission by up to an order of magnitude without increasing excitation power. PA-RICS and CC-RICS will enable improved quantification of the binding kinetic constants of DNA-PKCS variants at DNA damage sites. Importantly, the proposed RICS techniques can potentially be used to quantify the kinetics of a wide range of DNA damage sensing, signaling, and repair proteins with sparse accumulation patterns in the nucleus. Therefore, the proposed methods are very generally applicable to the DNA repair field and beyond. PUBLIC HEALTH RELEVANCE: We propose to develop fluorescence microscopy techniques that will enable researchers to monitor if and how fast cells can fix DNA after this is damaged by X-rays or chemical agents. By looking at how cancer cells respond to treatment-induced DNA damage when their repair proteins work properly, or when they don't due to a genetic mutation, we can understand the mechanisms of treatment resistance and design better therapies.

描述（由申请人提供）：大多数荧光标记的DNA修复蛋白的动力学在暴露于电离辐射或放射性化学剂之后，如果没有严重研究该系统正在研究的情况下，就无法在活细胞中量化活细胞中。除了少数几个在DNA损伤位点附近的蛋白质（例如3-H2AX，53BP1）外，大多数其他蛋白质附着在DNA损伤位点附近的较少副本中附着，无法通过荧光显微镜进行可视化。这是因为自由移动或不动的荧光蛋白具有较高的背景，这些蛋白会掩盖DNA修复蛋白在损伤位点的弱聚集。结果，通常通过用激光束照明诱导聚类的DNA损伤来可视化DNA损伤。这种照明诱导了核中一个或多个大斑点的修复蛋白的高积累。然而，激光诱导的损伤本质上是复杂的，可能不是用于在体内研究DNA修复的电离辐射或放射性映射的良好替代物。在这项工作中，我们建议开发定量显微镜方法，该方法可以克服当前无法量化荧光标记的DNA修复蛋白动力学的主要局限性。更具体地说，我们将应用栅格图像相关光谱（RICS），该技术分析图像像素中的时空荧光强度波动，以量化细胞核中稀疏积累的蛋白质修复动力学。我们将使用RIC来量化DNA依赖性蛋白激酶催化亚基（DNA-PKC）的修复动力学，其野生型和缺乏修复的7A形式在暴露于三射线和博霉素之后，它们都是双链断裂的代理。我们还将测试过氧化氢（单链断裂（DSB）形成剂）作为阴性对照。我们将表明，DSB形成后的DNA-PKCS动力学可以通过RIC量化。此外，我们建议开发两种专业形式的RIC，以进一步增强这些蛋白质的DNA修复动力学的量化：（i）光激活RIC（PA-RICS）将控制荧光修复蛋白浓度，（II）相干控制RICS（CC-RICS（CC）将通过增强激光效率增强效率的效率增强的效果，以增强量的效果。 PA-rics和CC-rics将能够改善DNA损伤位点DNA-PKCS变体的结合动力学常数的定量。重要的是，提出的RICS技术可以潜在地用于量化各种DNA损伤感应，信号传导和修复蛋白的动力学，并具有稀疏的积累模式。因此，所提出的方法通常非常适用于DNA修复场及其他地区。 公共卫生相关性：我们建议开发荧光显微镜技术，以使研究人员能够监测X射线或化学剂破坏此后的DNA是否可以修复DNA的速度。通过查看癌细胞对治疗诱导的DNA损伤的反应，当它们的修复蛋白正常工作时，或者由于基因突变而不作出反应，我们可以理解治疗耐药性和设计更好疗法的机制。