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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

基本信息

批准号：
8305487
负责人：
Georgios Alexandrakis
金额：
$ 14.31万
依托单位：
UNIVERSITY OF TEXAS ARLINGTON
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-08-01 至 2014-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8305487
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

项目摘要

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.

描述（由申请人提供）：暴露于电离辐射或拟放射性化学试剂后，大多数荧光标记的DNA修复蛋白的动力学在活细胞中无法定量，除非研究系统受到严重干扰。除了少数蛋白质大量附着在DNA损伤位点附近（如3-H2 AX，53 BP 1），大多数其他蛋白质附着在DNA损伤位点附近的拷贝数较少，并且不能通过荧光显微镜观察到。这是因为自由移动或固定的荧光蛋白的高背景掩盖了DNA修复蛋白在损伤部位的弱聚集。因此，DNA损伤通常通过用激光束照射诱导成簇的DNA损伤来可视化。这种光照诱导修复蛋白在细胞核中的一个或多个大斑点中非常高的积累。然而，激光诱导的损伤在本质上是复杂的，并且可能不是用于研究体内DNA修复的电离辐射或拟辐射剂的良好替代物。在这项工作中，我们建议开发定量显微镜方法，可以克服目前的主要限制，不能量化的动力学荧光标记的DNA修复蛋白在稀疏的DNA损伤位点。更具体地说，我们将应用光栅图像相关光谱（RICS），一种分析图像像素中的时空荧光强度波动的技术，以量化细胞核中稀疏积累的蛋白质的修复动力学。我们将使用RICS来量化DNA依赖性蛋白激酶催化亚基（DNA-PKCS）的修复动力学，在其野生型和修复缺陷的7A形式暴露于3-射线和博莱霉素后，这两个双链断裂形成剂。我们还将检测过氧化氢（一种单链断裂（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RICS定量。此外，我们建议开发两种专门形式的RICS，以进一步增强这些蛋白质的DNA修复动力学的定量：（i）光活化RICS（PA-RICS）将提供对荧光标记的修复蛋白浓度的控制，以及（ii）相干控制RICS（CC-RICS）将优化激光脉冲特性，以在不增加激发功率的情况下将荧光发射增强高达一个数量级。PA-RICS和CC-RICS将能够改进DNA损伤位点处DNA-PKCS变体的结合动力学常数的定量。重要的是，所提出的RICS技术可以潜在地用于量化细胞核中具有稀疏积累模式的广泛的DNA损伤传感、信号传导和修复蛋白的动力学。因此，所提出的方法非常普遍地适用于DNA修复领域和其他领域。