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修复蛋白在暴露于电离辐射或拟辐射化学试剂后，如果不对正在研究的系统进行严重扰动，则无法在活细胞中进行量化。除了少数蛋白质大量附着在DNA损伤部位(如3-H2AX、53BP1)外，大多数其他蛋白质在DNA损伤部位附近附着的拷贝数较少，无法在荧光显微镜下观察到。这是因为自由移动或不移动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损伤通常是通过激光照射诱导成簇的DNA损伤来可视化的。这种光照诱导修复蛋白在细胞核中的一个或多个大点上高度积累。然而，激光诱导的损伤本质上是复杂的，可能不是研究体内DNA修复的电离辐射或拟辐射试剂的良好替代品。在这项工作中，我们建议开发定量显微镜方法，以克服目前无法量化稀疏DNA损伤位置上荧光标记的DNA修复蛋白的动力学的主要限制。更具体地说，我们将应用栅格图像相关光谱(RICS)，这是一种分析图像像素中时空荧光强度波动的技术，以量化在细胞核中稀疏积累的蛋白质的修复动力学。我们将使用RICS来定量DNA依赖的蛋白激酶催化亚基(DNA-PKCS)，其野生型和修复缺陷的7A形式的修复动力学，暴露于三射线和博莱霉素，这两个都是双链断裂的形成因素。我们还将测试过氧化氢，一种单链断裂(DSB)形成剂，作为阴性对照。我们将证明DNA-PKCS在双链断裂形成后的动力学可以用RICS来定量。此外，我们建议开发两种特殊形式的RICS来进一步提高这些蛋白质的DNA修复动力学的量化：(I)光激活RICS(PA-RICS)将提供对荧光标记修复蛋白浓度的控制，(Ii)相干控制RICS(CC-RICS)将优化激光脉冲特性，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将改进DNA-PKCS变异体在DNA损伤部位的结合动力学常数的量化。重要的是，所提出的RICS技术可能被用来量化广泛的DNA损伤传感、信号和修复蛋白质的动力学，这些蛋白质在细胞核中具有稀疏的积累模式。因此，所提出的方法非常普遍地适用于DNA修复领域及以后的领域。 与公共健康相关：我们建议开发荧光显微镜技术，使研究人员能够监测细胞在受到X射线或化学试剂破坏后是否以及以多快的速度修复DNA。通过观察癌细胞在修复蛋白正常工作时或由于基因突变而不工作时，对治疗诱导的DNA损伤有何反应，我们可以了解治疗耐药的机制，并设计更好的治疗方法。